Lipase Variants and Polynucleotides Encoding Same

ABSTRACT

The present invention relates to a lipase variant of a parent lipase, which variant has lipase activity, at least 75% but less than 100% sequence identity to SEQ ID NO: 3 and comprises a substitution at one or more positions corresponding to positions 1; 2; 3; 4; 5; 6; 7; 9; 10; 11; 12; 16; 19; 30; 31; 34; 36; 37; 39; 40; 42; 44; 51; 52; 53; 54; 56; 58; 59; 70; 71; 72; 73; 83; 84; 86; 88; 90; 92; 93; 95; 96; 100; 101; 102; 104; 106; 109; 110; 112; 117; 119; 124; 125; 127; 128; 131; 132; 133; 134; 135; 137; 158; 159; 160; 161; 162; 163; 165; 166; 167; 168; 170; 181; 182; 183; 189; 190; 192; 194; 196; 202; 210; 211; 212; 220; 225; 227; 228; 229; 230; 231; 233; 237; 238; 239; 240; 242; 246; 247; 248; 252; 259; 262; 264; 269 of SEQ ID NO: 3. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to lipase variants, polynucleotidesencoding the variants, methods of producing the variants, and methods ofusing the variants.

BACKGROUND OF THE INVENTION

Lipases are important biocatalysts which have shown to be useful forvarious applications. Lipases have been employed in the removal of lipidstains and have been added to various compositions. Current cleaningand/or fabric care compositions comprise many active ingredients whichare interfering with the ability of lipases to remove lipid stains.Thus, the need exists for lipases that can function in the harshenvironment of compositions used for cleaning.

SUMMARY OF THE INVENTION

The present invention provides lipase variants with improved propertiesas compared to its parent, in particular lipase variants useful forcleaning. The variants are achieved by introducing modifications such assubstitutions into a parent lipase. The improved properties may be oneor more selected from: increase in thermostability; increase inthermostability in detergent; increased in hydrolytic activity indetergent; and increase in stability in detergent. The present inventionprovides lipase variants including but not limited to variants that areparticularly useful in various cleaning applications. The invention alsoprovides methods of cleaning using the lipase variants of the presentinvention.

In one embodiment the present invention relates to a lipase variant of aparent lipase, which variant has lipase activity, at least 75% but lessthan 100% sequence identity to SEQ ID NO: 3 and comprises a substitutionat one or more positions corresponding to positions 1; 2; 3; 4; 5; 6; 7;9; 10; 11; 12; 16; 19; 30; 31; 34; 36; 37; 39; 40; 42; 44; 51; 52; 53;54; 56; 58; 59; 70; 71; 72; 73; 83; 88; 92; 93; 95; 96; 100; 101; 102;104; 106; 109; 110; 112; 117; 119; 124; 125; 127; 128; 131; 132; 133;134; 135; 137; 158; 159; 160; 161; 162; 163; 165; 166; 167; 168; 170;181; 182; 183; 189; 190; 192; 194; 196; 202; 210; 211; 212; 220; 225;227; 228; 229; 230; 231; 233; 237; 238; 239; 240; 242; 246; 247; 248;252; 259; 262; 264; 269 of SEQ ID NO: 3.

The present invention also relates to polynucleotides encoding thevariants; nucleic acid constructs, vectors, and host cells comprisingthe polynucleotides; and methods of producing and use of the variants.

DETAILED DISCLOSURE OF THE INVENTION Definitions

Lipase: The terms “lipase”, “lipase enzyme”, “lipolytic enzyme”, “lipidesterase”, “lipolytic polypeptide”, and “lipolytic protein” refers to anenzyme in class EC3.1.1 as defined by Enzyme Nomenclature. It may havelipase activity (triacylglycerol lipase, EC3.1.1.3), cutinase activity(EC3.1.1.74), sterol esterase activity (EC3.1.1.13) and/or wax-esterhydrolase activity (EC3.1.1.50). For purposes of the present invention,lipase activity is determined according to the procedure described inthe Examples. In one aspect, the variants of the present invention haveat least 20%, e.g., at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or 100% of the lipase activity of the polypeptide ofSEQ ID NO: 3.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a variant. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding avariant of the present invention. Each control sequence may be native(i.e., from the same gene) or foreign (i.e., from a different gene) tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Expression: The term “expression” includes any step involved in theproduction of a variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has lipaseactivity. In one aspect, a fragment contains at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% but less than 100% of thenumber of amino acids 1 to 269 of SEQ ID NO: 3.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5× SSPE, 0.3% SDS, 200 microgram/mL shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2× SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Improved property: The term “improved property” means a characteristicassociated with a variant that is improved compared to the parent. Suchimproved properties include, but are not limited to: Stability such ase.g. thermostability, thermostability in detergent, stability indetergent; Activity such as e.g. hydrolytic activity, hydrolyticactivity in detergent; Substrate specific activity; Stain removal suchas e.g. lipid stain removal; and Wash performance.

Isolated: The term “isolated” means a substance in a form or environmentwhich does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5× SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2× SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 1 to 269 of SEQ ID NO: 2 which is identicalwith amino acids 1 to 269 of SEQ ID NO: 3. It is known in the art that ahost cell may produce a mixture of two or more different maturepolypeptides (i.e., with a different C-terminal and/or N-terminal aminoacid) expressed by the same polynucleotide. Accordingly, in one aspectthe mature peptide may start at amino acid 1, 2, 3, 4, or 5 of SEQ IDNO: 3.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving lipase activity. In one aspect, the mature polypeptide codingsequence is nucleotides 292 to 1098 of SEQ ID NO: 1.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5× SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2× SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5× SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2× SSC, 0.2% SDS at 60° C.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent or parent lipase: The term “parent” or “parent lipase” means alipase to which an alteration is made to produce the enzyme variants ofthe present invention. The parent may be a naturally occurring(wild-type) polypeptide or a variant or fragment thereof.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the -nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having lipase activity. In one aspect, a subsequence containsat least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% butless than 100% of the number of nucleotides 292 to 1098 of SEQ ID NO: 1.

Variant: The term “variant” means a polypeptide having lipase activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion, at one or more (e.g., several) positions. A substitution meansreplacement of the amino acid occupying a position with a differentamino acid; a deletion means removal of the amino acid occupying aposition; and an insertion means adding an amino acid adjacent to andimmediately following the amino acid occupying a position. The variantsof the present invention have at least 20%, e.g., at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or 100% of the lipase activity of SEQ ID NO: 3.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5× SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2× SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5× SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2× SSC, 0.2% SDS at 45° C.

Wild-type lipase: The term “wild-type” lipase means a lipase expressedby a naturally occurring microorganism, such as a bacterium, yeast, orfilamentous fungus found in nature.

Conventions for Designation of Variants

For purposes of the present invention, the polypeptide disclosed in SEQID NO: 3 is used to determine the corresponding amino acid residue inanother lipase. The amino acid sequence of another lipase is alignedwith SEQ ID NO: 3, and based on the alignment, the amino acid positionnumber corresponding to any amino acid residue in SEQ ID NO: 3 isdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofthe EMBOSS package (EMBOSS: The European Molecular Biology Open SoftwareSuite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version5.0.0 or later. The parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix.

Identification of the corresponding amino acid residue in another lipasecan be determined by an alignment of multiple polypeptide sequencesusing several computer programs including, but not limited to, MUSCLE(multiple sequence comparison by log-expectation; version 3.5 or later;Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066;Katoh et al., 2005, Nucleic Acids Research 33: 511-518; Katoh and Toh,2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods inMolecular Biology 537:_39-64; Katoh and Toh, 2010, Bioinformatics26:_1899-1900), and EMBOSS EMMA employing ClustalW (1.83 or later;Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), usingtheir respective default parameters.

When the other enzyme has diverged from the polypeptide of SEQ ID NO: 3such that traditional sequence-based comparison fails to detect theirrelationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615),other pairwise sequence comparison algorithms can be used. Greatersensitivity in sequence-based searching can be attained using searchprograms that utilize probabilistic representations of polypeptidefamilies (profiles) to search databases. For example, the PSI-BLASTprogram generates profiles through an iterative database search processand is capable of detecting remote homologs (Atschul et al., 1997,Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can beachieved if the family or superfamily for the polypeptide has one ormore representatives in the protein structure databases. Programs suchas GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin andJones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviation is employed.

Substitutions. For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of threonine at position 226 withalanine is designated as “Thr226Ala” or “T226A”. Multiple mutations areseparated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or“G205R+S411F”, representing substitutions at positions 205 and 411 ofglycine (G) with arginine (R) and serine (S) with phenylalanine (F),respectively.

Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofglycine at position 195 is designated as “Gly195*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*”or “G195*+S411*”.

Insertions. For an amino acid insertion, the following nomenclature isused: Original amino acid, position, original amino acid, inserted aminoacid. Accordingly the insertion of lysine after glycine at position 195is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Multiple alterations. Variants comprising multiple alterations areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of arginine and glycine atpositions 170 and 195 with tyrosine and glutamic acid, respectively.

Different alterations. Where different alterations can be introduced ata position, the different alterations are separated by a comma, e.g.,“Arg170Tyr,Glu” represents a substitution of arginine at position 170with tyrosine or glutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala”designates the following variants: “Tyr167Gly+Arg170Gly”,“Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

Variants

The present invention relates to a lipase variant of a parent lipase,which variant has lipase activity, at least 75% but less than 100%sequence identity to SEQ ID NO: 3 and comprises a substitution at one ormore positions corresponding to positions 1; 2; 3; 4; 5; 6; 7; 9; 10;11; 12; 16; 19; 30; 31; 34; 36; 37; 39; 40; 42; 44; 51; 52; 53; 54; 56;58; 59; 70; 71; 72; 73; 83; 88; 92; 93; 95; 96; 100; 101; 102; 104; 106;109; 110; 112; 117; 119; 124; 125; 127; 128; 131; 132; 133; 134; 135;137; 158; 159; 160; 161; 162; 163; 165; 166; 167; 168; 170; 181; 182;183; 189; 190; 192; 194; 196; 202; 210; 211; 212; 220; 225; 227; 228;229; 230; 231; 233; 237; 238; 239; 240; 242; 246; 247; 248; 252; 259;262; 264; 269 of SEQ ID NO: 3.

The present invention also relates to a variant, wherein the variantcomprises a substitution at one or more residues corresponding topositions 1(C,F,G,H,I,L,M,P,Q,R,V,W,Y); 2(A,C,D,M,N,P,Q,R,S,T,V);3(A,E,G,K,Q,S,T,V); 4(A,P,Q,R,S,T); 5(E,K,S,T,V,Y);6(A,H,K,N,P,Q,R,S,V,W); 7(P); 8(C,G,H,N,P,Q,R,S,W,Y); 9(Y); 10(G,R,S,Y);11(G,H,I,L,P,Q,R,T); 12(A,I,K,L,M,N,R,S,T); 16(P,R,S,T,W);19(C,E,H,L,W,T); 30(A,F,H,I,L,N,P,S,T,V,W);31(A,C,D,E,F,G,H,I,K,P,Q,R,S,V); 34(A,G,V); 36(D,E,G,K,Q,R,S,V);37(A,E,G,S,V); 39(F,K,L,N,S); 40(A,D,E,G,I,L,R,V);41(A,D,E,G,H,K,L,P,Q,R,S,V,W); 42(A,E,G,K,L,M,R,S,T,V);44(A,C,E,G,H,K,M,P,Q,R,S,V,W); 51(A,D,G,H,K,L,M,V,Y); 52(L,V);53(A,D,G,H,I,L,M,P,Q,R,S,V); 54(A,D,E,G,I,K,L,S,V); 56(A,P,Q,R,T,V,W);58(C,G,I,P,R,S,V,W); 59(A,D,F,G,H,L,M,P,R,S,T,V); 61(G,M); 70(A,E,G,R);71(E,K,N,Q,R,W,Y); 72(A,C,D,G,K,R,S,T,V); 73(E,G,L,N,Q,R,S,T); 83(A);84(A,E,L,P,W,Y); 86(A,G,I,K,M,W); 88(M,Q,R,S);90(D,F,G,L,S,T,V,W);91(E,F,G,Q,Y); 92(A,C,D,F,P,S,T,V,W); 93(A,I,R,V);95(C,G,I,Q,R,S); 96(A,C,D,E,G,N,Q,R,S,W,Y); 98(A,D,E,G,K,N,R,T,V,W);100(A,E,G,K,L,R,S,V,W); 101(D,H,R,S,V); 102(H,I,P,R,S);103(A,C,D,F,G,L,P,V); 104(A,C,D,E,K,L,M,P,Q,R,V,W,Y);106(A,C,G,L,P,Q,R,S,T,W); 109(A,D,E,G,H,L,N,P,Q,R,S,T); 110(C,P,S,V);112(F,H,Q,R,W); 113(M,W); 116(A,C,D,F,R,S,T);117(A,D,G,N,P,R,S,V);119(D,E,F,I,R,S,T,V); 120(A,E,G,H,L,M,S,T,V,Y);124(C,D,G,H,L,M,Q,R,S,V,W); 125(A,D,E,G,I,K,L,M,Q,R,S,V);127(A,F,G,H,I,Q,R,S,V,Y); 128(A,E,G,H,M,Q,S,T); 131(A,G,I,M,S,T,V,W);132(G,S,V); 133(A,E,K,L,M,R,V,W); 134(A,D,E,G,K,R,S,T,V);135(A,C,G,P,Q,R,T,V,W,Y); 137(A,F,G,P,R); 158(A,C,G,I,L,N,R,S,T,V,W);159(G,T); 160(A,D,G,I,L,Q,V); 161(C,G,I,K,L,P,Q,R,S);162(A,D,G,L,M,P,R,S,V,W); 163(A,D,E,H,L,M,Q,V,Y);165(A,C,G,K,L,M,P,R,V,W); 166(A,C,E,G,L,P,R,W); 167(C,F,H,I,L,M,R,V);168(A,C,D,G,R,S,V,W); 170(A,C,E,G,H,I,K,L,M,Q,R,S,V,W,Y);181(A,C,E,G,L,Q,R,S,V,W); 182(A,C,F,G,I,L,R,S,T,V,W,Y);183(E,V);186(A,C,E,G,H,K,L,R,S,T,W); 189(A,C,G,L,M,P,R,S);190(A,E,G,I,K,N,R,V,W); 192(A,I,L,M,P,R,T,V); 194(A,D,E,F,G,H,L,Q,T,V);196(A,Q,Y); 201(D,G,L,P); 202(I,M); 204(E,K,L,P,R); 208(E,M,P,R,W);210(A,F,L,Q); 211(C,E,G,P,S,V,W); 212(D,E,G,H,K,N,P,Q,R,S,T,V,W);213(E,H,L,M,P,Q,R,W); 220(A,C,D,G,L,P,R,S,T,W); 225(A,L,M,P,Q,S);226(C,E,F,G,P,R,W,Y); 227(A,C,G,P,R,S,V,W); 228(A,C,D,G,L,P,R,V,W);229(A,I,L,N,R,S,T); 230(A,G,H,L,P,Q,R,S,T,V,W);231(A,C,G,H,K,R,S,V,W,Y); 233(A,F,H,L,M,S,T); 236(C,L,P,S,V);237(F,G,H,L,M,N,R,V); 238(A,E,F,G,H,M,P,R,S,V,W); 239(F,G,S,Y); 240(A);241(M,V); 242(A,C,G,I,L,M,P,R,T,V,Y); 243(A,C,E,F,G,M);246(A,E,K,P,S,T,V); 247(A,E,L,P,T,Y); 248(A,G,P,V);252(A,D,E,G,N,Q,S,V); 255(C,E,M,Q,R);259(A,C,D,E,G,I,K,L,M,Q,R,T,V,W,Y); 262(A,D,E,K,M,R,T,V);264(C,G,I,L,M,P,R,S,T); 267(E,H,K,M,Q,W);269(A,D,E,G,H,K,L,M,N,Q,R,S,V,Y) of SEQ ID NO: 3.

In some aspects the invention relates to a variant selected from thegroup consisting of: (a) a polypeptide having at least 80%, at least82%, at least 84%, at least 86%, at least 88%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% but less than 100%sequence identity to the polypeptide of SEQ ID NO: 3; (b) a polypeptideencoded by a polynucleotide that hybridizes under low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the polypeptide coding sequence of SEQ ID NO: 1 or(ii) the full-length complement of (i); (c) a polypeptide encoded by apolynucleotide having at least 80%, at least 82%, at least 84%, at least86%, at least 88%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% but less than 100% sequence identity to thepolypeptide coding sequence of SEQ ID NO: 3; and (d) a fragment of thepolypeptide of SEQ ID NO: 3, wherein the fragment has lipase activity.

In some aspects the invention relates to a variant which has sequenceidentity of at least 80%, at least 82%, at least 84%, at least 86%, atleast 88%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% but less than 100% sequence identity to the amino acidsequence of the parent lipase.

In some aspects the invention relates to a variant which has at least atleast 80%, at least 82%, at least 84%, at least 86%, at least 88%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% butless than 100% sequence identity to SEQ ID NO: 3.

In some aspects the invention relates to a variant wherein the number ofsubstitutions in the variants of the present invention is 1-40, e.g.,1-30, 1-20, 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 substitutions.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position 51. In anotheraspect, the amino acid at a position corresponding to position 51 issubstituted with C,F,G,H,I,L,M,P,Q,R,V,W,Y. In another aspect, thevariant comprises or consists of the substitutionS1(C,F,G,H,I,L,M,P,Q,R,V,W,Y) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position 12. In anotheraspect, the amino acid at a position corresponding to position 12 issubstituted with A,C,D,M,N,P,Q,R,S,T,V. In another aspect, the variantcomprises or consists of the substitution 12(A,C,D,M,N,P,Q,R,S,T,V) ofSEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position D3. In anotheraspect, the amino acid at a position corresponding to position D3 issubstituted with A,E,G,K,Q,S,T,V. In another aspect, the variantcomprises or consists of the substitution D3(A,E,G,K,Q,S,T,V) of SEQ IDNO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position G4. In anotheraspect, the amino acid at a position corresponding to position G4 issubstituted with A,P,Q,R,S,T. In another aspect, the variant comprisesor consists of the substitution G4(A,P,Q,R,S,T) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position G5. In anotheraspect, the amino acid at a position corresponding to position G5 issubstituted with E,K,S,T,V,Y. In another aspect, the variant comprisesor consists of the substitution G5(E,K,S,T,V,Y) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position I6. In anotheraspect, the amino acid at a position corresponding to position I6 issubstituted with A,H,K,N,P,Q,R,S,V,W. In another aspect, the variantcomprises or consists of the substitution I6(A,H,K,N,P,Q,R,S,V,W) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position I6. In anotheraspect, the amino acid at a position corresponding to position I6 issubstituted with A,H,K,N,P,Q,R,S,V,W. In another aspect, the variantcomprises or consists of the substitution I6(A,H,K,N,P,Q,R,S,V,W) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position R7. In anotheraspect, the amino acid at a position corresponding to position R7 issubstituted with P. In another aspect, the variant comprises or consistsof the substitution R7(P) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position A8. In anotheraspect, the amino acid at a position corresponding to position A8 issubstituted with C,G,H,N,P,Q,R,S,W,Y. In another aspect, the variantcomprises or consists of the substitution A8(C,G,H,N,P,Q,R,S,W,Y) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position A9. In anotheraspect, the amino acid at a position corresponding to position A9 issubstituted with Y. In another aspect, the variant comprises or consistsof the substitution A8(Y) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position T10. Inanother aspect, the amino acid at a position corresponding to positionT10 is substituted with G,R,S,Y. In another aspect, the variantcomprises or consists of the substitution T10(G,R,S,Y) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position S11. Inanother aspect, the amino acid at a position corresponding to positionS11 is substituted with G,H,I,L,P,Q,R,T. In another aspect, the variantcomprises or consists of the substitution S11(G,H,I,L,P,Q,R,T) of SEQ IDNO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position Q12. Inanother aspect, the amino acid at a position corresponding to positionQ12 is substituted with A,I,K,L,M,N,R,S,T. In another aspect, thevariant comprises or consists of the substitution Q12(A,I,K,L,M,N,R,S,T)of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position E16. Inanother aspect, the amino acid at a position corresponding to positionE16 is substituted with P,R,S,T,W. In another aspect, the variantcomprises or consists of the substitution E16(P,R,S,T,W) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position Y19. Inanother aspect, the amino acid at a position corresponding to positionY19 is substituted with C,E,H,L,W,T. In another aspect, the variantcomprises or consists of the substitution Y19(C,E,H,L,W,T) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position R30. Inanother aspect, the amino acid at a position corresponding to positionR30 is substituted with A,F,H,I,L,N,P,S,T,V,W. In another aspect, thevariant comprises or consists of the substitutionR30(A,F,H,I,L,N,P,S,T,V,W) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position T31. Inanother aspect, the amino acid at a position corresponding to positionT31 is substituted with A,C,D,E,F,G,H,I,K,P,Q,R,S,V. In another aspect,the variant comprises or consists of the substitutionT31(A,C,D,E,F,G,H,I,K,P,Q,R,S,V) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position P34. Inanother aspect, the amino acid at a position corresponding to positionP34 is substituted with A,G,V. In another aspect, the variant comprisesor consists of the substitution P34(A,G,V) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position A36. Inanother aspect, the amino acid at a position corresponding to positionA36 is substituted with D,E,G,K,Q,R,S,V. In another aspect, the variantcomprises or consists of the substitution A36(D,E,G,K,Q,R,S,V) of SEQ IDNO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position T37. Inanother aspect, the amino acid at a position corresponding to positionT37 is substituted with A,E,G,S,V. In another aspect, the variantcomprises or consists of the substitution T37(A,E,G,S,V) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position D39. Inanother aspect, the amino acid at a position corresponding to positionD39 is substituted with F,K,L,N,S. In another aspect, the variantcomprises or consists of the substitution D39(F,K,L,N,S) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position C40. Inanother aspect, the amino acid at a position corresponding to positionC40 is substituted with A,D,E,G,I,L,R,V. In another aspect, the variantcomprises or consists of the substitution C40(A,D,E,G,I,L,R,V) of SEQ IDNO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position 141. Inanother aspect, the amino acid at a position corresponding to position141 is substituted with A,D,E,G,H,K,L,P,Q,R,S,V,W. In another aspect,the variant comprises or consists of the substitutionI41(A,D,E,G,H,K,L,P,Q,R,S,V,W) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position H42. Inanother aspect, the amino acid at a position corresponding to positionH42 is substituted with A,E,G,K,L,M,R,S,T,V. In another aspect, thevariant comprises or consists of the substitutionH42(A,E,G,K,L,M,R,S,T,V) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position D44. Inanother aspect, the amino acid at a position corresponding to positionD44 is substituted with A,C,E,G,H,K,M,P,Q,R,S,V,W. In another aspect,the variant comprises or consists of the substitutionD44(A,C,E,G,H,K,M,P,Q,R,S,V,W) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position I51. Inanother aspect, the amino acid at a position corresponding to positionI51 is substituted with A,D,G,H,K,L,M,V,Y. In another aspect, thevariant comprises or consists of the substitution I51(A,D,G,H,K,L,M,V,Y)of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position I52. Inanother aspect, the amino acid at a position corresponding to positionI52 is substituted with L,V. In another aspect, the variant comprises orconsists of the substitution I52(L,V) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position K53. Inanother aspect, the amino acid at a position corresponding to positionK53 is substituted with A,D,G,H,I,L,M,P,Q,R,S,V. In another aspect, thevariant comprises or consists of the substitutionK53(A,D,G,H,I,L,M,P,Q,R,S,V) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position T54. Inanother aspect, the amino acid at a position corresponding to positionT54 is substituted with A,D,E,G,I,K,L,S,V. In another aspect, thevariant comprises or consists of the substitution T54(A,D,E,G,I,K,L,S,V)of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position S56. Inanother aspect, the amino acid at a position corresponding to positionS56 is substituted with A,P,Q,R,T,V,W. In another aspect, the variantcomprises or consists of the substitution S56(A,P,Q,R,T,V,W) of SEQ IDNO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position L58. Inanother aspect, the amino acid at a position corresponding to positionL58 is substituted with C,G,I,P,R,S,V,W. In another aspect, the variantcomprises or consists of the substitution L58(C,G,I,P,R,S,V,W) of SEQ IDNO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position I59. Inanother aspect, the amino acid at a position corresponding to positionI59 is substituted with A,D,F,G,H,L,M,P,R,S,T,V. In another aspect, thevariant comprises or consists of the substitutionI59(A,D,F,G,H,L,M,P,R,S,T,V) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position D61. Inanother aspect, the amino acid at a position corresponding to positionD61 is substituted with G,M. In another aspect, the variant comprises orconsists of the substitution D61(G,M) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position D70. Inanother aspect, the amino acid at a position corresponding to positionD70 is substituted with A,E,G,R. In another aspect, the variantcomprises or consists of the substitution D70(A,E,G,R) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position S71. Inanother aspect, the amino acid at a position corresponding to positionS71 is substituted with E,K,N,Q,R,W,Y. In another aspect, the variantcomprises or consists of the substitution S71(E,K,N,Q,R,W,Y) of SEQ IDNO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position E72. Inanother aspect, the amino acid at a position corresponding to positionE72 is substituted with A,C,D,G,K,R,S,T,V. In another aspect, thevariant comprises or consists of the substitution E72(A,C,D,G,K,R,S,T,V)of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position K73. Inanother aspect, the amino acid at a position corresponding to positionK73 is substituted with E,G,L,N,Q,R,S,T. In another aspect, the variantcomprises or consists of the substitution K73(E,G,L,N,Q,R,S,T) of SEQ IDNO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position S83. Inanother aspect, the amino acid at a position corresponding to positionS83 is substituted with A. In another aspect, the variant comprises orconsists of the substitution S83(A) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position S84. Inanother aspect, the amino acid at a position corresponding to positionS84 is substituted with A,E,L,P,W,Y. In another aspect, the variantcomprises or consists of the substitution S84(A,E,L,P,W,Y) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position R86. Inanother aspect, the amino acid at a position corresponding to positionR86 is substituted with A,G,I,K,M,W. In another aspect, the variantcomprises or consists of the substitution R86(A,G,I,K,M,W) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position W88. Inanother aspect, the amino acid at a position corresponding to positionW88 is substituted with M,Q,R,S. In another aspect, the variantcomprises or consists of the substitution W88(M,Q,R,S) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position A90. Inanother aspect, the amino acid at a position corresponding to positionA90 is substituted with D,F,G,L,S,T,V,W). In another aspect, the variantcomprises or consists of the substitution A90(D,F,G,L,S,T,V,W) of SEQ IDNO: 3. In some aspects the invention relates to a variant comprises orconsists of a substitution at a position corresponding to position D91.In another aspect, the amino acid at a position corresponding toposition D91 is substituted with E,F,G,Q,Y. In another aspect, thevariant comprises or consists of the substitution D91(E,F,G,Q,Y) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position L92. Inanother aspect, the amino acid at a position corresponding to positionL92 is substituted with A,C,D,F,P,S,T,V,W. In another aspect, thevariant comprises or consists of the substitution L92(A,C,D,F,P,S,T,V,W)of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position T93. Inanother aspect, the amino acid at a position corresponding to positionT93 is substituted with A,I,R,V. In another aspect, the variantcomprises or consists of the substitution T93(A,I,R,V) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position V95. Inanother aspect, the amino acid at a position corresponding to positionV95 is substituted with C,G,I,Q,R,S. In another aspect, the variantcomprises or consists of the substitution V95(C,G,I,Q,R,S) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position P96. Inanother aspect, the amino acid at a position corresponding to positionP96 is substituted with A,C,D,E,G,N,Q,R,S,W,Y. In another aspect, thevariant comprises or consists of the substitutionP96(A,C,D,E,G,N,Q,R,S,W,Y) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position S98. Inanother aspect, the amino acid at a position corresponding to positionS98 is substituted with A,D,E,G,K,N,R,T,V,W. In another aspect, thevariant comprises or consists of the substitutionS98(A,D,E,G,K,N,R,T,V,W) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position P100. Inanother aspect, the amino acid at a position corresponding to positionP100 is substituted with A,E,G,K,L,R,S,V,W. In another aspect, thevariant comprises or consists of the substitutionP100(A,E,G,K,L,R,S,V,W) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position P101. Inanother aspect, the amino acid at a position corresponding to positionP101 is substituted with D,H,R,S,V. In another aspect, the variantcomprises or consists of the substitution P101(D,H,R,S,V) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position V102. Inanother aspect, the amino acid at a position corresponding to positionV102 is substituted with H,I,P,R,S. In another aspect, the variantcomprises or consists of the substitution V102(H,I,P,R,S) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position S103. Inanother aspect, the amino acid at a position corresponding to positionS103 is substituted with A,C,D,F,G,L,P,V. In another aspect, the variantcomprises or consists of the substitution 5103(A,C,D,F,G,L,P,V) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position G104. Inanother aspect, the amino acid at a position corresponding to positionG104 is substituted with A,C,D,E,K,L,M,P,Q,R,V,W,Y. In another aspect,the variant comprises or consists of the substitutionG104(A,C,D,E,K,L,M,P,Q,R,V,W,Y) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position K106. Inanother aspect, the amino acid at a position corresponding to positionK106 is substituted with A,C,G,L,P,Q,R,S,T,W. In another aspect, thevariant comprises or consists of the substitutionK106(A,C,G,L,P,Q,R,S,T,W) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position K109. Inanother aspect, the amino acid at a position corresponding to positionK109 is substituted with A,D,E,G,H,L,N,P,Q,R,S,T. In another aspect, thevariant comprises or consists of the substitutionK109(A,D,E,G,H,L,N,P,Q,R,S,T) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position G110. Inanother aspect, the amino acid at a position corresponding to positionG110 is substituted with C,P,S,V. In another aspect, the variantcomprises or consists of the substitution G110 (C,P,S,V) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position L112. Inanother aspect, the amino acid at a position corresponding to positionL112 is substituted with F,H,Q,R,W. In another aspect, the variantcomprises or consists of the substitution L112 (F,H,Q,R,W) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position D113. Inanother aspect, the amino acid at a position corresponding to positionD113 is substituted with M,W. In another aspect, the variant comprisesor consists of the substitution D113 (M,W) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position G116. Inanother aspect, the amino acid at a position corresponding to positionG116 is substituted with A,C,D,F,R,S,T. In another aspect, the variantcomprises or consists of the substitution G116 (A,C,D,F,R,S,T) of SEQ IDNO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position E117. Inanother aspect, the amino acid at a position corresponding to positionE117 is substituted with A,D,G,N,P,R,S,V. In another aspect, the variantcomprises or consists of the substitution E117 (A,D,G,N,P,R,S,V) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position Q119. Inanother aspect, the amino acid at a position corresponding to positionQ119 is substituted with D,E,F,I,R,S,T,V. In another aspect, the variantcomprises or consists of the substitution Q119 (D,E,F,I,R,S,T,V) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position N120. Inanother aspect, the amino acid at a position corresponding to positionN120 is substituted with A,E,G,H,L,M,S,T,V,Y. In another aspect, thevariant comprises or consists of the substitution N120(A,E,G,H,L,M,S,T,V,Y) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position A124. Inanother aspect, the amino acid at a position corresponding to positionA124 is substituted with C,D,G,H,L,M,Q,R,S,V,W. In another aspect, thevariant comprises or consists of the substitution A124(C,D,G,H,L,M,Q,R,S,V,W) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position T125. Inanother aspect, the amino acid at a position corresponding to positionT125 is substituted with A,D,E,G,I,K,L,M,Q,R,S,V. In another aspect, thevariant comprises or consists of the substitution T125(A,D,E,G,I,K,L,M,Q,R,S,V) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position L127. Inanother aspect, the amino acid at a position corresponding to positionL127 is substituted with A,F,G,H,I,Q,R,S,V,Y. In another aspect, thevariant comprises or consists of the substitution L127(A,F,G,H,I,Q,R,S,V,Y) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position D128. Inanother aspect, the amino acid at a position corresponding to positionD128 is substituted with A,E,G,H,M,Q,S,T. In another aspect, the variantcomprises or consists of the substitution D128 (A,E,G,H,M,Q,S,T) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position K131. Inanother aspect, the amino acid at a position corresponding to positionK131 is substituted with A,G,I,M,S,T,V,W. In another aspect, the variantcomprises or consists of the substitution K131 (A,G,I,M,S,T,V,W) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position Q132. Inanother aspect, the amino acid at a position corresponding to positionQ132 is substituted with G,S,V. In another aspect, the variant comprisesor consists of the substitution Q132 (G,S,V) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position Y133. Inanother aspect, the amino acid at a position corresponding to positionY133 is substituted with A,E,K,L,M,R,V,W. In another aspect, the variantcomprises or consists of the substitution Y133 (A,E,K,L,M,R,V,W) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position P134. Inanother aspect, the amino acid at a position corresponding to positionP134 is substituted with A,D,E,G,K,R,S,T,V. In another aspect, thevariant comprises or consists of the substitution P134(A,D,E,G,K,R,S,T,V) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position S135. Inanother aspect, the amino acid at a position corresponding to positionS135 is substituted with A,C,G,P,Q,R,T,V,W,Y. In another aspect, thevariant comprises or consists of the substitution S135(A,C,G,P,Q,R,T,V,W,Y) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position K137. Inanother aspect, the amino acid at a position corresponding to positionK137 is substituted with A,F,G,P,R. In another aspect, the variantcomprises or consists of the substitution K137 (A,F,G,P,R) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position Y158. Inanother aspect, the amino acid at a position corresponding to positionY158 is substituted with A,C,G,I,L,N,R,S,T,V,W. In another aspect, thevariant comprises or consists of the substitution Y158(A,C,G,I,L,N,R,S,T,V,W) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position Q159. Inanother aspect, the amino acid at a position corresponding to positionQ159 is substituted with G,T. In another aspect, the variant comprisesor consists of the substitution Q159 (G,T) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position R160. Inanother aspect, the amino acid at a position corresponding to positionR160 is substituted with A,D,G,I,L,Q,V. In another aspect, the variantcomprises or consists of the substitution R160 (A,D,G,I,L,Q,V) of SEQ IDNO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position E161. Inanother aspect, the amino acid at a position corresponding to positionE161 is substituted with C,G,I,K,L,P,Q,R,S. In another aspect, thevariant comprises or consists of the substitution E161(C,G,I,K,L,P,Q,R,S) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position E162. Inanother aspect, the amino acid at a position corresponding to positionE162 is substituted with A,D,G,L,M,P,R,S,V,W. In another aspect, thevariant comprises or consists of the substitution E162(A,D,G,L,M,P,R,S,V,W) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position G163. Inanother aspect, the amino acid at a position corresponding to positionG163 is substituted with A,D,E,H,L,M,Q,V,Y. In another aspect, thevariant comprises or consists of the substitution G163(A,D,E,H,L,M,Q,V,Y) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position S165. Inanother aspect, the amino acid at a position corresponding to positionS165 is substituted with A,C,G,K,L,M,P,R,V,W. In another aspect, thevariant comprises or consists of the substitution S165(A,C,G,K,L,M,P,R,V,W) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position S166. Inanother aspect, the amino acid at a position corresponding to positionS166 is substituted with A,C,E,G,L,P,R,W. In another aspect, the variantcomprises or consists of the substitution S166 (A,C,E,G,L,P,R,W) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position S167. Inanother aspect, the amino acid at a position corresponding to positionS167 is substituted with C,F,H,I,L,M,R,V. In another aspect, the variantcomprises or consists of the substitution S167 (C,F,H,I,L,M,R,V) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position N168. Inanother aspect, the amino acid at a position corresponding to positionN168 is substituted with A,C,D,G,R,S,V,W. In another aspect, the variantcomprises or consists of the substitution N168 (A,C,D,G,R,S,V,W) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position F170. Inanother aspect, the amino acid at a position corresponding to positionF170 is substituted with A,C,E,G,H,I,K,L,M,Q,R,S,V,W,Y. In anotheraspect, the variant comprises or consists of the substitution F170(A,C,E,G,H,I,K,L,M,Q,R,S,V,W,Y) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position D181. Inanother aspect, the amino acid at a position corresponding to positionD181 is substituted with A,C,E,G,L,Q,R,S,V,W. In another aspect, thevariant comprises or consists of the substitution D181(A,C,E,G,L,Q,R,S,V,W) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position P182. Inanother aspect, the amino acid at a position corresponding to positionP182 is substituted with A,C,F,G,I,L,R,S,T,V,W,Y. In another aspect, thevariant comprises or consists of the substitution P182(A,C,F,G,I,L,R,S,T,V,W,Y) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position A183. Inanother aspect, the amino acid at a position corresponding to positionA183 is substituted with E,V. In another aspect, the variant comprisesor consists of the substitution A183 (E,V) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position N186. Inanother aspect, the amino acid at a position corresponding to positionN186 is substituted with A,C,E,G,H,K,L,R,S,T,W. In another aspect, thevariant comprises or consists of the substitution N186(A,C,E,G,H,K,L,R,S,T,W) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position V189. Inanother aspect, the amino acid at a position corresponding to positionV189 is substituted with A,C,G,L,M,P,R,S. In another aspect, the variantcomprises or consists of the substitution V189 (A,C,G,L,M,P,R,S) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position S190. Inanother aspect, the amino acid at a position corresponding to positionS190 is substituted with A,E,G,I,K,N,R,V,W. In another aspect, thevariant comprises or consists of the substitution S190(A,E,G,I,K,N,R,V,W) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position G192. Inanother aspect, the amino acid at a position corresponding to positionG192 is substituted with A,I,L,M,P,R,T,V. In another aspect, the variantcomprises or consists of the substitution G192 (A,I,L,M,P,R,T,V) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position P194. Inanother aspect, the amino acid at a position corresponding to positionP194 is substituted with A,D,E,F,G,H,L,Q,T,V. In another aspect, thevariant comprises or consists of the substitution P194(A,D,E,F,G,H,L,Q,T,V) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position R196. Inanother aspect, the amino acid at a position corresponding to positionR196 is substituted with A,Q,Y. In another aspect, the variant comprisesor consists of the substitution R196 (A,Q,Y) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position E201. Inanother aspect, the amino acid at a position corresponding to positionE201 is substituted with D,G,L,P. In another aspect, the variantcomprises or consists of the substitution E201 (D,G,L,P) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position R202. Inanother aspect, the amino acid at a position corresponding to positionR202 is substituted with I,M. In another aspect, the variant comprisesor consists of the substitution R202 (I,M) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position I204. Inanother aspect, the amino acid at a position corresponding to positionI204 is substituted with E,K,L,P,R. In another aspect, the variantcomprises or consists of the substitution I204 (E,K,L,P,R) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position L208. Inanother aspect, the amino acid at a position corresponding to positionL208 is substituted with E,M,P,R,W. In another aspect, the variantcomprises or consists of the substitution L208 (E,M,P,R,W) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position P210. Inanother aspect, the amino acid at a position corresponding to positionP210 is substituted with A,F,L,Q. In another aspect, the variantcomprises or consists of the substitution P210 (A,F,L,Q) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position A211. Inanother aspect, the amino acid at a position corresponding to positionA211 is substituted with C,E,G,P,S,V,W. In another aspect, the variantcomprises or consists of the substitution A211 (C,E,G,P,S,V,W) of SEQ IDNO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position A212. Inanother aspect, the amino acid at a position corresponding to positionA212 is substituted with D,E,G,H,K,N,P,Q,R,S,T,V,W. In another aspect,the variant comprises or consists of the substitution A212(D,E,G,H,K,N,P,Q,R,S,T,V,W) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position F213. Inanother aspect, the amino acid at a position corresponding to positionF213 is substituted with E,H,L,M,P,Q,R,W. In another aspect, the variantcomprises or consists of the substitution F213 (E,H,L,M,P,Q,R,W) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position E220. Inanother aspect, the amino acid at a position corresponding to positionE220 is substituted with A,C,D,G,L,P,R,S,T,W. In another aspect, thevariant comprises or consists of the substitution E220(A,C,D,G,L,P,R,S,T,W) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position T225. Inanother aspect, the amino acid at a position corresponding to positionT225 is substituted with A,L,M,P,Q,S. In another aspect, the variantcomprises or consists of the substitution T225 (A,L,M,P,Q,S) of SEQ IDNO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position D226. Inanother aspect, the amino acid at a position corresponding to positionD226 is substituted with C,E,F,G,P,R,W,Y. In another aspect, the variantcomprises or consists of the substitution D226 (C,E,F,G,P,R,W,Y) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position N227. Inanother aspect, the amino acid at a position corresponding to positionN227 is substituted with A,C,G,P,R,S,V,W. In another aspect, the variantcomprises or consists of the substitution N227 (A,C,G,P,R,S,V,W) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position S228. Inanother aspect, the amino acid at a position corresponding to positionS228 is substituted with A,C,D,G,L,P,R,V,W. In another aspect, thevariant comprises or consists of the substitution S228(A,C,D,G,L,P,R,V,W) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position P229. Inanother aspect, the amino acid at a position corresponding to positionP229 is substituted with A,I,L,N,R,S,T. In another aspect, the variantcomprises or consists of the substitution P229 (A,I,L,N,R,S,T) of SEQ IDNO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position E230. Inanother aspect, the amino acid at a position corresponding to positionE230 is substituted with A,G,H,L,P,Q,R,S,T,V,W. In another aspect, thevariant comprises or consists of the substitution E230(A,G,H,L,P,Q,R,S,T,V,W) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position T231. Inanother aspect, the amino acid at a position corresponding to positionT231 is substituted with A,C,G,H,K,R,S,V,W,Y. In another aspect, thevariant comprises or consists of the substitution T231(A,C,G,H,K,R,S,V,W,Y) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position Q233. Inanother aspect, the amino acid at a position corresponding to positionQ233 is substituted with A,F,H,L,M,S,T. In another aspect, the variantcomprises or consists of the substitution Q233 (A,F,H,L,M,S,T) of SEQ IDNO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position T236. Inanother aspect, the amino acid at a position corresponding to positionT236 is substituted with C,L,P,S,V. In another aspect, the variantcomprises or consists of the substitution T236 (C,L,P,S,V) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position S237. Inanother aspect, the amino acid at a position corresponding to positionS237 is substituted with F,G,H,L,M,N,R,V. In another aspect, the variantcomprises or consists of the substitution S237 (F,G,H,L,M,N,R,V) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position D238. Inanother aspect, the amino acid at a position corresponding to positionD238 is substituted with A,E,F,G,H,M,P,R,S,V,W. In another aspect, thevariant comprises or consists of the substitution D238(A,E,F,G,H,M,P,R,S,V,W) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position L239. Inanother aspect, the amino acid at a position corresponding to positionL239 is substituted with F,G,S,Y. In another aspect, the variantcomprises or consists of the substitution L239 (F,G,S,Y) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position E240. Inanother aspect, the amino acid at a position corresponding to positionE240 is substituted with A. In another aspect, the variant comprises orconsists of the substitution E240 (A) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position T241. Inanother aspect, the amino acid at a position corresponding to positionT241 is substituted with M,V. In another aspect, the variant comprisesor consists of the substitution T241 (M,V) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position S242. Inanother aspect, the amino acid at a position corresponding to positionS242 is substituted with A,C,G,I,L,M,P,R,T,V,Y. In another aspect, thevariant comprises or consists of the substitution S242(A,C,G,I,L,M,P,R,T,V,Y) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position D243. Inanother aspect, the amino acid at a position corresponding to positionD243 is substituted with A,C,E,F,G,M. In another aspect, the variantcomprises or consists of the substitution D243 (A,C,E,F,G,M) of SEQ IDNO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position N246. Inanother aspect, the amino acid at a position corresponding to positionN246 is substituted with A,E,K,P,S,T,V. In another aspect, the variantcomprises or consists of the substitution N246 (A,E,K,P,S,T,V) of SEQ IDNO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position S247. Inanother aspect, the amino acid at a position corresponding to positionS247 is substituted with A,E,L,P,T,Y. In another aspect, the variantcomprises or consists of the substitution S247 (A,E,L,P,T,Y) of SEQ IDNO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position I248. Inanother aspect, the amino acid at a position corresponding to positionI248 is substituted with A,G,P,V. In another aspect, the variantcomprises or consists of the substitution I248 (A,G,P,V) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position T252. Inanother aspect, the amino acid at a position corresponding to positionT252 is substituted with A,D,E,G,N,Q,S,V. In another aspect, the variantcomprises or consists of the substitution T252 (A,D,E,G,N,Q,S,V) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position L255. Inanother aspect, the amino acid at a position corresponding to positionL255 is substituted with C,E,M,Q,R. In another aspect, the variantcomprises or consists of the substitution L255 (C,E,M,Q,R) of SEQ ID NO:3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position S259. Inanother aspect, the amino acid at a position corresponding to positionS259 is substituted with A,C,D,E,G,I,K,L,M,Q,R,T,V,W,Y. In anotheraspect, the variant comprises or consists of the substitution S259(A,C,D,E,G,I,K,L,M,Q,R,T,V,W,Y) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position G262. Inanother aspect, the amino acid at a position corresponding to positionG262 is substituted with A,D,E,K,M,R,T,V. In another aspect, the variantcomprises or consists of the substitution G262 (A,D,E,K,M,R,T,V) of SEQID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position N264. Inanother aspect, the amino acid at a position corresponding to positionN264 is substituted with C,G,I,L,M,P,R,S,T. In another aspect, thevariant comprises or consists of the substitution N264(C,G,I,L,M,P,R,S,T) of SEQ ID NO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position L267. Inanother aspect, the amino acid at a position corresponding to positionL267 is substituted with E,H,K,M,Q,W. In another aspect, the variantcomprises or consists of the substitution L267 (E,H,K,M,Q,W) of SEQ IDNO: 3.

In some aspects the invention relates to a variant comprises or consistsof a substitution at a position corresponding to position T269. Inanother aspect, the amino acid at a position corresponding to positionT269 is substituted with A,D,E,G,H,K,L,M,N,Q,R,S,V,Y. In another aspect,the variant comprises or consists of the substitution T269(A,D,E,G,H,K,L,M,N,Q,R,S,V,Y) of SEQ ID NO: 3.

In some aspects the invention related to a variant, wherein the varianthas one or more (e.g several) improved property relative to the parentmeasured as an Improvement Factor (IF) that is greater that 1.0, whereinthe improved property is selected from the group consisting of:Thermostability; Thermostability in Detergent; Hydrolytic Activity inDetergent; Stability in Detergent.

In some aspects the invention related to a variant, wherein theImprovement Factor (IF) is greater that 1.0 in at least one of theassays selected from the group consisting of: A Thermostability Assay; BThermostability in Detergent Assay; C Performance Screening Assay; and DStability in Detergent Assay.

In some aspects the invention related to a variant, wherein theImprovement Factor (IF) is greater that 1.0 in at least two, in at leastthree, or in at least four assays.

In some aspects the invention related to a variant, wherein theImprovement Factor (IF) is at least 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7;1.8; 1.9; 2.0; 2.1; 2.3; 2.4; or 2.5.

Amino acid positions within a molecule that are useful for makingvariants are those positions wherein at least one substitution leads toa variant exhibiting an improved characteristic as compared to theunchanged molecule i.e. parent i.e. IF>1.0. The improved characteristicmay be determined using the assay protocols described in example 1: AThermostability Assay; B Thermostability in Detegent Assay; C Detergentstability Assay; or D Performance screening Assay.

In some aspects the invention relates to a variant, wherein the varianthas lipase activity and comprises a substitution at one or more (e.g.several) of the positions corresponding to position 1; 2; 3; 4; 5; 6;11; 12; 16; 30; 31; 37; 39; 40; 42; 44; 51; 53; 54; 58; 59; 71; 72; 73;88; 92; 93; 95; 96; 102; 104; 106; 109; 110; 112; 117; 119; 124; 125;127; 128; 131; 132; 134; 135; 137; 158; 160; 162; 163; 165; 166; 167;168; 170; 181; 182; 190; 194; 196; 202; 210; 212; 220; 225; 227; 228;229; 230; 231; 233; 237; 238; 239; 242; 246; 259; 264; 269 of SEQ ID NO:3, or of a polypeptide having at least 75%, at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identity to SEQ ID NO: 3 and has improved thermostability ascompared to the parent i.e. IF>1.0. The improved property may bemeasured by “A Thermostability Assay” as described in Example 1.

In some aspects the invention relates to a variant, wherein the varianthas lipase activity and comprises one or more (e.g. several) of thesubstitutions: 1(C,F,G,H,I,L,M,P,Q,R,V,W,Y); 2(C,M,N,P,R,T,V);3(A,E,G,K,T,V); 4(A,P,Q,R,S,T); 5(E,K,Y); 6(H,K,P,Q,R); 8(C,P,R,S,W);11(I,L,P,R); 12(A,I,L,M,R,S,T); 16(P,R,W); 30(L,P,S); 31(F,Q,V); 37(A);39(N); 40(E,I,L); 41(A,D,E,G,H,L,P,Q,R,S,V,W); 42(A,E,G,L,M,R,T,V);44(A,C,G,K,M,P,R,V,W); 51(A,G,K,L,M,V); 53(A,D,G,H,I,L,M,Q,R,S,V);54(A,D,E,G,I,K,L,S,V); 58(C,G,P,R,W); 59(A,H,M,R,S,T,V); 71(K,R);72(A,C,D,G,K,R,S,V); 73(E,G,L,N,Q,R); 84(A,E,L,P,W,Y); 88(M,Q,R);90(D,G,S,T,V,W); 91(G,Q,Y); 92(A,D,F,S,T,V); 93(A,R,V); 95(C,G,I,Q,R,S);96(C,D,E,G,N,Q,S,W,Y); 98(A,D,E,G); 102(H,I,R); 103(A,C,D,F,G,P);104(K,L,R); 106(L,P,Q,R); 109(A,E,R,S); 110(C,V); 112(F,Q,W);117(A,R,S); 119(E,R,S); 124(G,L,S,W); 125(A,M,R,S,V); 127(A,F,H,R,S);128(A,E,Q,T); 131(G,M,T); 132(G,V); 134(A,D,E,G,K,R,S,T,V);135(A,G,Q,V,W,Y); 137(A,P,R); 158(W); 160(A,D,G,I,Q,V);162(A,G,L,M,P,R,S,V,W); 163(A,D,E,Q); 165(A,G,K,R); 166(A,G,P);167(H,I,L); 168(A,C,G,R,S,V,W); 170(A,E,G,H,I,K,L,M,R,V,Y); 181(E,W);182(F,I,R,Y); 186(A,C,E,G,K,R,S); 190(A,E,G,K,N,R,W); 194(A,D,E,H,L,T);196(A,Q,Y); 201(D,G,L,P); 202(I,M); 204(E,K,L,P,R); 208(E,M,R,W);210(A); 212(D,E,G,H,K,N,P,Q,R,S,T,V,W); 213(E,H,L,M,Q,R,W);220(A,C,G,L,P,R,S,T); 225(L,M); 226(C,F,G,P,R,W,Y); 227(S); 228(P,R,V);229(A,L,N,R,S,T); 230(G,H,P,R,S,V,W); 231(A,C,G,H,K,R,S,V,Y);233(A,F,H,L,M,S); 236(V); 237(F,G,H,R); 238(A,E,F,G,H,M,P,R,S,V,W);239(F,Y); 242(A,C,P,T); 243(A,C,F,G,M); 246(P); 255(C,E,M,Q,R);259(C,E,L,M,R,T,V); 264(C,P,R,S); 267(E); 269(D,E,G,L,M,Q,R,V) of SEQ IDNO: 3, or of a polypeptide having at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% identity to SEQ ID NO: 3 and has improved thermostabilityas compared to the parent i.e. IF>1.0. The improved property may bemeasured by “A Thermostability Assay” as described in Example 1.

In some aspects the invention relates to a variant, wherein the varianthas lipase activity and comprises a substitution at one or more (e.g.several) of the positions corresponding to position 1; 2; 3; 4; 5; 6; 7;10; 11; 16; 19; 30; 31; 36; 42; 44; 51; 52; 54; 56; 58; 59; 70; 71; 72;73; 88; 92; 93; 96; 100; 101; 104; 106; 109; 110; 112; 119; 124; 125;127; 128; 131; 133; 134; 135; 158; 160; 161; 162; 163; 165; 166; 167;170; 181; 182; 189; 190; 192; 194; 210; 211; 212; 220; 225; 227; 228;229; 230; 231; 233; 237; 238; 239; 242; 246; 247; 248; 252; 259; 264;269 of SEQ ID NO: 3, or of a polypeptide having at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identity to SEQ ID NO: 3 and has improvedthermostability in detergent as compared to the parent i.e. IF>1.0. Theimproved property may be measured by “B Thermostability in DetergentAssay” as described in Example 1.

In some aspects the invention relates to a variant, wherein the varianthas lipase activity and comprises one or more (e.g. several) of thesubstitutions: 1(C,F,G,H,I,M,Q,R,V,W,Y); 2(A,C,D,N,P,Q,R,S,T,V);3(E,G,Q,S); 4(A,P,S,T); 5(S,T,V,Y); 6(A,H,K,N,P,Q,R,S,V,W); 7(P);8(C,G,H,N,P,Q,R,S,W,Y); 10(S,Y); 11(H,P,Q,R,T); 16(S,T); 19(C,E,H,L,W);30(A,F,H,L,P,S,T,W); 31(C,D,E,G,I,K,Q,S,V); 36(E,G,K,Q);42(G,K,L,R,S,V); 44(A,E,G,H,K,P,Q,S,V); 51(V); 52(L,V); 54(A,I);56(Q,T); 58(C,G,I,P,R,W); 59(D,F,H,L,T,V); 70(A,E,G,R);71(E,K,N,Q,R,W,Y); 72(A,T,V); 73(E,G,N,R,T); 86(G,I,K,M); 88(R,S);90(F); 91(F,Y); 92(A,S,V); 93(A,I); 96(A,C,D,E,G,N,Q,R,W,Y);98(A,E,G,K,N,T,V,W); 100(A,E,K,L,R,V,W); 101(D,H,R,S,V); 103(A,D,G,L,V);104(A,C,D,E,K,L,M,P,Q,R,V,W,Y); 106(A,C,L,P,T);109(A,D,E,G,H,L,Q,R,S,T); 110(C,S,V); 112(F,H,Q,R,W); 116(A,S,T);119(D,E,F,R,S,T,V); 120(A,E,G,H,L,M,S,T,V,Y);124(C,D,G,H,L,M,Q,R,S,V,W); 125(A,D,G,K,M,R,S,V); 127(F,G,H,I,R,S,V,Y);128(A,G,Q,T); 131(A,G,I,M,S,T,V,W); 133(A,E,K,L,R,W);134(A,D,E,G,K,R,S,T); 135(A,C,G,P,R,W,Y); 158(C,G,I,L,N,R,S,T,V);160(V); 161(C,G,I,L,Q,R,S); 162(A,D,L,M,P,R,S,V); 163(A,D,E,H,L,M,Q,V);165(C,G,K,L,M,P,R,V,W); 166(A,C,G,P,W); 167(C,F,I,L,M,R,V); 170(A,C);181(A,G,L,Q,R,S,V,W); 182(A,C,F,G,I,L,R,S,T,V,W,Y);186(A,E,G,H,L,R,S,T,W); 189(A,C,G,L,M,P,R,S); 190(A,E,G,I,K,R,V);192(A,I,L,M,P,R,T,V); 194(E,G,Q); 201(D,G); 204(K,P); 208(M); 210(L);211(G,P,S,V,W); 212(D,E,G,H,K,N,R,S); 213(E,H,L,M,P,R,W);220(D,G,R,S,W); 225(A,L,P,S); 226(C,E,G,P,W); 227(C,G,P,R,S,V);228(A,G,L,R); 229(I,L,N,S); 230(A,G,H,L,P,Q,R,S,T,W); 231(C,G,H,K,R,S);233(A,S,T); 236(C,L,P,S,V); 237(F,L,M,N,R,V); 238(A,E,G,P,R,S,V);239(S); 241(M,V); 242(A,I,L,M,P,R,T,V,Y); 243(M); 246(A,K,S,T);247(A,E,L,T,Y); 248(V); 252(A,E,G,N,S,V); 255(C,M,Q);259(A,C,D,E,M,Q,T,V,W,Y); 264(G,I,L,M,P,S,T); 267(E,H,K,M,Q,W);269(A,E,G,H,K,L,M,N,Q,R,S,V,Y) of SEQ ID NO: 3, or of a polypeptidehaving at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQID NO: 3 and has improved thermostability in detergent as compared tothe parent i.e. IF>1.0. The improved property may be measured by “BThermostability in Detergent Assay” as described in Example 1.

In some aspects the invention relates to a variant, wherein the varianthas lipase activity and comprises a substitution at one or more (e.g.several) of the positions corresponding to position 1; 2; 5; 6; 9; 10;11; 12; 19; 30; 31; 34; 36; 37; 39; 40; 42; 44; 51; 52; 53; 54; 56; 58;59; 70; 71; 72; 73; 83; 92; 93; 95; 96; 100; 102; 104; 106; 109; 110;112; 117; 119; 124; 125; 127; 128; 131; 132; 133; 134; 135; 137; 158;159; 160; 161; 162; 163; 165; 166; 167; 168; 170; 181; 182; 183; 189;190; 192; 194; 210; 211; 212; 220; 225; 227; 228; 229; 230; 231; 238;239; 240; 242; 246; 247; 248; 252; 259; 262; 264 of SEQ ID NO: 3, or ofa polypeptide having at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 3 and has improved hydrolytic activity indetergent as compared to the parent i.e. IF>1.0. The improved propertymay be measured by “C Performance Screening Assay” as described inExample 1.

In some aspects the invention relates to a variant, wherein the varianthas lipase activity and comprises one or more (e.g. several) of thesubstitutions: 1(M); 2(Q,T); 5(S); 6(K,N,V); 9(Y); 10(G,R,S);11(G,H,I,L,P,R); 12(I,K,L,M,N,R,S,T); 19(C,T); 30(A,F,I,L,N,P,T,V);31(A,G,H,P,R); 34(A,G,V); 36(D,E,G,K,R,S,V); 37(A,E,G,S,V); 39(F,K,L,S);40(A,E,G,I,L,V); 41(A,H,K,L,P,R,V); 42(A,R,V); 44(G,P); 51(A,D,G,H,V,Y);52(L,V); 53(G,H,L,P,Q,R); 54(K,V); 56(A,P,Q,R,T,V,W); 58(C,S,V);59(D,G,P,R,V); 61(G,M); 70(A,G); 71(N,W); 72(A,G,K,R,T); 73(E,G,Q,R,S);83(A); 86(A,G,W); 90(F,L); 91(E); 92(C,P,V,W); 93(A,V); 95(G,R);96(C,G,S,Y); 98(A,D,R,V); 100(A,G,R,S,W); 102(P,R,S); 103(A,G,L,P,V);104(L,M,R); 106(A,G,S,W); 109(L,N,P,R); 110(P,S); 112(Q,R); 113(M,W);116(C,D,F,R); 117(A,D,G,N,P,R,V); 119(E,I,R,S,T); 120(M);124(L,M,Q,R,S,V); 125(A,D,E,G,I,L,M,Q,R,S,V); 127(Q,V); 128(E,Q,T);131(A,V); 132(S); 133(E,M,V,W); 134(A,D,G,K,R); 135(G,R,T,Y);158(C,G,I,N,R,S); 159(G,T); 160(A,L,Q,V); 161(C,K,L,P,Q,R);162(A,G,P,S,W); 163(E,L,Q,Y); 165(A,L,R,V,W); 166(A,C,E,G,L,R); 167(H);168(A,D); 170(C); 181(C,E); 182(A,C,G,I,L,R,S,T,V,W,Y); 183(E,V);186(G,S); 189(G,L,P,R); 190(A,E,G,K,R,V); 192(P,T); 194(A,F,G,L,V);201(G,L); 208(P,R,W); 210(F,Q); 211(C,E,G,S,V,W); 212(D,G,H,P,Q,S,T);213(M,Q,R); 220(A,C,D,G,P,S); 225(L,Q); 226(C,E,G,R,W); 227(A,C,G,W);228(A,C,D,G,R,V,W); 229(L); 230(G,P,R,S); 231(A,G,H,R,W,Y);238(A,G,H,V); 239(G,S); 240(A); 241(V); 242(A,G,P,R,T,V); 243(A,E,M);246(A,E,P,S,V); 247(L,P); 248(A,G,P); 252(A,D,G,N,Q,S,V);259(A,G,I,K,L,M,Q,R,V,Y); 262(A,D,E,K,M,R,T,V); 264(I,P,R) of SEQ ID NO:3, or of a polypeptide having at least 75%, at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identity to SEQ ID NO: 3 and has improved hydrolytic activity indetergent as compared to the parent i.e. IF>1.0. The improved propertymay be measured by “C Performance Screening Assay” as described inExample 1.

In some aspects the invention relates to a variant, wherein the varianthas lipase activity and comprises a substitution at one or more (e.g.several) of the positions corresponding to position 2; 3; 4; 5; 6; 7;16; 42; 44; 54; 71; 92; 93; 96; 106; 109; 112; 127; 131; 133; 135; 162;163; 166; 192; 202; 220; 227; 228; 230; 238; 247; 259; 264 of SEQ ID NO:3, or of a polypeptide having at least 75%, at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identity to SEQ ID NO: 3 and has improved stability in detergent ascompared to the parent i.e. IF>1.0. The improved property may bemeasured by “D Stability in Detergent Assay” as described in Example 1.

In some aspects the invention relates to a variant, wherein the varianthas lipase activity and comprises one or more (e.g. several) of thesubstitutions: 2(A,D,Q,S); 3(E,K); 4(A,P,Q,R,S); 5(K,T,Y); 6(K,P); 7(P);10(S); 16(T); 30(L); 31(E); 36(R); 42(K,L,R); 44(A,G,H,K,S); 54(L,V);71(W,Y); 72(K); 92(F,S,T); 93(A,R,V); 96(A,C,Q,R,W); 98(E); 104(C,M,P);106(T); 109(R,S); 112(H,R,W); 119(D,R); 127(S); 131(A,G,I,M,S,T,V,W);133(A,E,W); 135(C,P,W); 158(I,S); 161(S); 162(G,L,P,R,S,V);163(A,D,E,M); 165(G); 166(A,C,G,W); 167(R,V); 182(I,T);192(I,L,M,R,T,V); 201(G,L); 213(E,L); 220(W); 226(C,G); 227(G,P,R,S);228(L,R); 229(R,S); 230(A,H,W); 231(R); 236(V); 238(A,P,S); 242(1);243(R); 247(T); 255(Q); 259(C,E,Q,V,W); 264(I,M,P,R,S); 267(H);269(A,G,L,M,N,Q) of SEQ ID NO: 3, or of a polypeptide having at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3 andhas improved stability in detergent as compared to the parent i.e.IF>1.0. The improved property may be measured by “D Stability inDetergent Assay” as described in Example 1.

The variants may further comprise one or more additional substitutionsat one or more (e.g., several) other positions.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, AlaNal, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

For example, the variants may further comprise a substitution at one ormore positions corresponding to: A8T; Y28LC; 141T; C43S; D48E; K50E;Y60AC; D61NKRAVLSTC; R80C; S84C; 186C; N87C; A90C; D91NKRAVLST; F94LTK;S98P; S103R; D113NKRAVLST; S114C; G116V; N120KD; K131F; G156D; N186Y;E201QKRAVLST; 1204VATSG; V205IL; L208VATSG; F213VATSG; H217N;D226NKRAVLST; T236A; T241S; D243NKRAVLSTH; V254IATSG; L255VATSG;D256NKRAVLST; L258VATSG; Y260WCGV; L267VATSG of SEQ ID NO: 3.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for lipase activity to identify amino acid residuesthat are critical to the activity of the molecule. See also, Hilton etal., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzymeor other biological interaction can also be determined by physicalanalysis of structure, as determined by such techniques as nuclearmagnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

In an embodiment, the variant has improved thermostability compared tothe parent enzyme. The thermostability may be determined by assay A asdescribed in example 1.

In an embodiment, the variant has improved thermostability in detergentcompared to the parent enzyme. The improved stability in detergent maybe determined by assay B as described in example 1.

In an embodiment, the variant has improved hydrolytic activity indetergent compared to the parent enzyme. The improved stability indetergent may be determined by assay C as described in example 1.

In an embodiment, the variant has improved stability in detergentcompared to the parent enzyme. The improved stability in detergent maybe determined by assay D as described in example 1.

Parent Lipases

The parent lipase may be (a) a polypeptide having at least 75% sequenceidentity to SEQ ID NO: 3; (b) a polypeptide encoded by a polynucleotidethat hybridizes under low stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the full-lengthcomplement of (i) or (ii); or (c) a polypeptide encoded by apolynucleotide having at least 75% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1.

In an aspect, the parent has a sequence identity to SEQ ID NO: 3 of atleast 75%, e.g., at least 80%, at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, which have lipaseactivity. In one aspect, the amino acid sequence of the parent differsby up to 40 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 from SEQ ID NO: 3.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 2. In another aspect, the parent comprises orconsists of the mature polypeptide of SEQ ID NO: 2. In another aspect,the parent comprises or consists of amino acids 1 to 269 of SEQ ID NO:2. In another aspect, the parent comprises or consists of SEQ ID NO: 3.In another aspect, the parent comprises or consists of amino acids 1 to269 of SEQ ID NO: 3.

In another aspect, the parent is a fragment of the polypeptide of SEQ IDNO: 3. In one aspect, a fragment contains at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% but less than 100% of thenumber of amino acids 1 to 269 of SEQ ID NO: 3.

In another embodiment, the parent is an allelic variant of SEQ ID NO: 3.

In another aspect, the parent is encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the full-length complement of (i) (Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 2 or a fragment thereof, may be used todesign nucleic acid probes to identify and clone DNA encoding a parentfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1 or a subsequence thereof, the carrier material is usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQID NO: 1; (iii) the full-length complement thereof; or (iv) asubsequence thereof; under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using, for example, X-ray film or any otherdetection means known in the art.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1. In another aspect, the nucleic acid probe isnucleotides 292 to 1098 of SEQ ID NO: 1. In another aspect, the nucleicacid probe is a polynucleotide that encodes the polypeptide of SEQ IDNO: 2; the mature polypeptide thereof; or a fragment thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 1.

In another embodiment, the parent is encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 of at least 60%, e.g., at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent may be obtained from microorganisms of any genus. Forpurposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a strain inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a filamentous fungal lipase such as an Acremonium,Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria,Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus,Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus,Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides,Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus,Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia,Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum,Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria lipase.

In another aspect, the parent is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonaturn, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa,Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris,Trichoderma harzianurn, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride lipase.

In another aspect, the parent is a Rhizomucor miehei lipase, e.g., thelipase of SEQ ID NO: 2 or the mature polypeptide thereof which isidentical with SEQ ID NO:3.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

The present invention also relates to methods for obtaining a lipasevariant and/or a fragment thereof, comprising introducing into a parentlipase a substitution at one or more positions corresponding topositions 1; 2; 3; 4; 5; 6; 7; 9; 10; 11; 12; 16; 19; 30; 31; 34; 36;37; 39; 40; 42; 44; 51; 52; 53; 54; 56; 58; 59; 70; 71; 72; 73; 83; 88;92; 93; 95; 96; 100; 101; 102; 104; 106; 109; 110; 112; 117; 119; 124;125; 127; 128; 131; 132; 133; 134; 135; 137; 158; 159; 160; 161; 162;163; 165; 166; 167; 168; 170; 181; 182; 183; 189; 190; 192; 194; 196;202; 210; 211; 212; 220; 225; 227; 228; 229; 230; 231; 233; 237; 238;239; 240; 242; 246; 247; 248; 252; 259; 262; 264; 269 of SEQ ID NO: 3,wherein the variant and/or fragment has lipase activity; and recoveringthe variant and/or fragment.

In some aspects the invention relates to methods for obtaining a lipasevariant and/or a fragment thereof, comprising introducing into a parentlipase a substitution at one or more positions corresponding topositions 1(C,F,G,H,I,L,M,P,Q,R,V,W,Y); 2(A,C,D,M,N,P,Q,R,S,T,V);3(A,E,G,K,Q,S,T,V); 4(A,P,Q,R,S,T); 5(E,K,S,T,V,Y);6(A,H,K,N,P,Q,R,S,V,W); 7(P); 8(C,G,H,N,P,Q,R,S,W,Y); 9(Y); 10(G,R,S,Y);11(G,H,I,L,P,Q,R,T); 12(A,I,K,L,M,N,R,S,T); 16(P,R,S,T,W);19(C,E,H,L,W,T); 30(A,F,H,I,L,N,P,S,T,V,W);31(A,C,D,E,F,G,H,I,K,P,Q,R,S,V); 34(A,G,V); 36(D,E,G,K,Q,R,S,V);37(A,E,G,S,V); 39(F,K,L,N,S); 40(A,D,E,G,I,L,R,V);41(A,D,E,G,H,K,L,P,Q,R,S,V,W); 42(A,E,G,K,L,M,R,S,T,V);44(A,C,E,G,H,K,M,P,Q,R,S,V,W); 51(A,D,G,H,K,L,M,V,Y); 52(L,V);53(A,D,G,H,I,L,M,P,Q,R,S,V); 54(A,D,E,G,I,K,L,S,V); 56(A,P,Q,R,T,V,W);58(C,G,I,P,R,S,V,W); 59(A,D,F,G,H,L,M,P,R,S,T,V); 61(G,M); 70(A,E,G,R);71(E,K,N,Q,R,W,Y); 72(A,C,D,G,K,R,S,T,V); 73(E,G,L,N,Q,R,S,T); 83(A);84(A,E,L,P,W,Y); 86(A,G,I,K,M,W); 88(M,Q,R,S);90(D,F,G,L,S,T,V,W);91(E,F,G,Q,Y); 92(A,C,D,F,P,S,T,V,W); 93(A,I,R,V);95(C,G,I,Q,R,S); 96(A,C,D,E,G,N,Q,R,S,W,Y); 98(A,D,E,G,K,N,R,T,V,W);100(A,E,G,K,L,R,S,V,W); 101(D,H,R,S,V); 102(H,I,P,R,S);103(A,C,D,F,G,L,P,V); 104(A,C,D,E,K,L,M,P,Q,R,V,W,Y);106(A,C,G,L,P,Q,R,S,T,W); 109(A,D,E,G,H,L,N,P,Q,R,S,T); 110(C,P,S,V);112(F,H,Q,R,W); 113(M,W); 116(A,C,D,F,R,S,T);117(A,D,G,N,P,R,S,V);119(D,E,F,I,R,S,T,V); 120(A,E,G,H,L,M,S,T,V,Y);124(C,D,G,H,L,M,Q,R,S,V,W); 125(A,D,E,G,I,K,L,M,Q,R,S,V);127(A,F,G,H,I,Q,R,S,V,Y); 128(A,E,G,H,M,Q,S,T); 131(A,G,I,M,S,T,V,W);132(G,S,V); 133(A,E,K,L,M,R,V,W); 134(A,D,E,G,K,R,S,T,V);135(A,C,G,P,Q,R,T,V,W,Y); 137(A,F,G,P,R); 158(A,C,G,I,L,N,R,S,T,V,W);159(G,T); 160(A,D,G,I,L,Q,V); 161(C,G,I,K,L,P,Q,R,S);162(A,D,G,L,M,P,R,S,V,W); 163(A,D,E,H,L,M,Q,V,Y);165(A,C,G,K,L,M,P,R,V,W); 166(A,C,E,G,L,P,R,W); 167(C,F,H,I,L,M,R,V);168(A,C,D,G,R,S,V,W); 170(A,C,E,G,H,I,K,L,M,Q,R,S,V,W,Y);181(A,C,E,G,L,Q,R,S,V,W); 182(A,C,F,G,I,L,R,S,T,V,W,Y);183(E,V);186(A,C,E,G,H,K,L,R,S,T,W); 189(A,C,G,L,M,P,R,S);190(A,E,G,I,K,N,R,V,W); 192(A,I,L,M,P,R,T,V); 194(A,D,E,F,G,H,L,Q,T,V);196(A,Q,Y); 201(D,G,L,P); 202(I,M); 204(E,K,L,P,R); 208(E,M,P,R,W);210(A,F,L,Q); 211(C,E,G,P,S,V,W); 212(D,E,G,H,K,N,P,Q,R,S,T,V,W);213(E,H,L,M,P,Q,R,W); 220(A,C,D,G,L,P,R,S,T,W); 225(A,L,M,P,Q,S);226(C,E,F,G,P,R,W,Y); 227(A,C,G,P,R,S,V,W); 228(A,C,D,G,L,P,R,V,W);229(A,I,L,N,R,S,T); 230(A,G,H,L,P,Q,R,S,T,V,W);231(A,C,G,H,K,R,S,V,W,Y); 233(A,F,H,L,M,S,T); 236(C,L,P,S,V);237(F,G,H,L,M,N,R,V); 238(A,E,F,G,H,M,P,R,S,V,W); 239(F,G,S,Y); 240(A);241(M,V); 242(A,C,G,I,L,M,P,R,T,V,Y); 243(A,C,E,F,G,M);246(A,E,K,P,S,T,V); 247(A,E,L,P,T,Y); 248(A,G,P,V);252(A,D,E,G,N,Q,S,V); 255(C,E,M,Q,R);259(A,C,D,E,G,I,K,L,M,Q,R,T,V,W,Y); 262(A,D,E,K,M,R,T,V);264(C,G,I,L,M,P,R,S,T); 267(E,H,K,M,Q,W);269(A,D,E,G,H,K,L,M,N,Q,R,S,V,Y) of SEQ ID NO: 3, wherein the variantand/or fragment has lipase activity; and recovering the variant and/orfragment.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO95/17413; or WO95/22625. Other methods that can be usedinclude error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to polynucleotides encoding a variantof the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO96/00787), Fusariumvenenatum amyloglucosidase (WO00/56900), Fusarium venenatum Daria(WO00/56900), Fusarium venenatum Quinn (WO00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellmay be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis ctyIIIA gene (WO94/25612) and a Bacillus subtilisSP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leadersequence is operably linked to the 5′-terminus of the polynucleotideencoding the variant. Any leader that is functional in the host cell maybe used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the variant-encoding sequence and,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the variant would be operably linkedwith the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. he vector preferably contains anelement(s) that permits integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMR1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thevariant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenaturn, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a variant,comprising: (a) cultivating a host cell of the present invention underconditions suitable for expression of the variant; and (b) recoveringthe variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants. These detection methods include, but are notlimited to, use of specific antibodies, formation of an enzyme product,or disappearance of an enzyme substrate. For example, an enzyme assaymay be used to determine the activity of the variant may be the assaydescribed in the examples below.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant.

Compositions.

In one aspect the invention relates to a composition comprising a lipasevariant of a parent lipase, which variant has lipase activity, at least75% but less than 100% sequence identity to SEQ ID NO: 3 and comprises asubstitution at one or more positions corresponding to positions 1; 2;3; 4; 5; 6; 7; 9; 10; 11; 12; 16; 19; 30; 31; 34; 36; 37; 39; 40; 42;44; 51; 52; 53; 54; 56; 58; 59; 70; 71; 72; 73; 83; 88; 92; 93; 95; 96;100; 101; 102; 104; 106; 109; 110; 112; 117; 119; 124; 125; 127; 128;131; 132; 133; 134; 135; 137; 158; 159; 160; 161; 162; 163; 165; 166;167; 168; 170; 181; 182; 183; 189; 190; 192; 194; 196; 202; 210; 211;212; 220; 225; 227; 228; 229; 230; 231; 233; 237; 238; 239; 240; 242;246; 247; 248; 252; 259; 262; 264; 269 of SEQ ID NO: 3.

In another aspect the invention relates to a composition comprising alipase variant of a parent lipase, which variant has lipase activity, atleast 75% but less than 100% sequence identity to SEQ ID NO: 3 andcomprises a substitution at one or more positions corresponding topositions 1(C,F,G,H,I,L,M,P,Q,R,V,W,Y); 2(A,C,D,M,N,P,Q,R,S,T,V);3(A,E,G,K,Q,S,T,V); 4(A,P,Q,R,S,T); 5(E,K,S,T,V,Y);6(A,H,K,N,P,Q,R,S,V,W); 7(P); 8(C,G,H,N,P,Q,R,S,W,Y); 9(Y); 10(G,R,S,Y);11(G,H,I,L,P,Q,R,T); 12(A,I,K,L,M,N,R,S,T); 16(P,R,S,T,W);19(C,E,H,L,W,T); 30(A,F,H,I,L,N,P,S,T,V,W);31(A,C,D,E,F,G,H,I,K,P,Q,R,S,V); 34(A,G,V); 36(D,E,G,K,Q,R,S,V);37(A,E,G,S,V); 39(F,K,L,N,S); 40(A,D,E,G,I,L,R,V);41(A,D,E,G,H,K,L,P,Q,R,S,V,W); 42(A,E,G,K,L,M,R,S,T,V);44(A,C,E,G,H,K,M,P,Q,R,S,V,W); 51(A,D,G,H,K,L,M,V,Y); 52(L,V);53(A,D,G,H,I,L,M,P,Q,R,S,V); 54(A,D,E,G,I,K,L,S,V); 56(A,P,Q,R,T,V,W);58(C,G,I,P,R,S,V,W); 59(A,D,F,G,H,L,M,P,R,S,T,V); 61(G,M); 70(A,E,G,R);71(E,K,N,Q,R,W,Y); 72(A,C,D,G,K,R,S,T,V); 73(E,G,L,N,Q,R,S,T); 83(A);84(A,E,L,P,W,Y); 86(A,G,I,K,M,W); 88(M,Q,R,S);90(D,F,G,L,S,T,V,W);91(E,F,G,Q,Y); 92(A,C,D,F,P,S,T,V,W); 93(A,I,R,V);95(C,G,I,Q,R,S); 96(A,C,D,E,G,N,Q,R,S,W,Y); 98(A,D,E,G,K,N,R,T,V,W);100(A,E,G,K,L,R,S,V,W); 101(D,H,R,S,V); 102(H,I,P,R,S);103(A,C,D,F,G,L,P,V); 104(A,C,D,E,K,L,M,P,Q,R,V,W,Y);106(A,C,G,L,P,Q,R,S,T,W); 109(A,D,E,G,H,L,N,P,Q,R,S,T); 110(C,P,S,V);112(F,H,Q,R,W); 113(M,W); 116(A,C,D,F,R,S,T);117(A,D,G,N,P,R,S,V);119(D,E,F,I,R,S,T,V); 120(A,E,G,H,L,M,S,T,V,Y);124(C,D,G,H,L,M,Q,R,S,V,W); 125(A,D,E,G,I,K,L,M,Q,R,S,V);127(A,F,G,H,I,Q,R,S,V,Y); 128(A,E,G,H,M,Q,S,T); 131(A,G,I,M,S,T,V,W);132(G,S,V); 133(A,E,K,L,M,R,V,W); 134(A,D,E,G,K,R,S,T,V);135(A,C,G,P,Q,R,T,V,W,Y); 137(A,F,G,P,R); 158(A,C,G,I,L,N,R,S,T,V,W);159(G,T); 160(A,D,G,I,L,Q,V); 161(C,G,I,K,L,P,Q,R,S);162(A,D,G,L,M,P,R,S,V,W); 163(A,D,E,H,L,M,Q,V,Y);165(A,C,G,K,L,M,P,R,V,W); 166(A,C,E,G,L,P,R,W); 167(C,F,H,I,L,M,R,V);168(A,C,D,G,R,S,V,W); 170(A,C,E,G,H,I,K,L,M,Q,R,S,V,W,Y);181(A,C,E,G,L,Q,R,S,V,W); 182(A,C,F,G,I,L,R,S,T,V,W,Y);183(E,V);186(A,C,E,G,H,K,L,R,S,T,W); 189(A,C,G,L,M,P,R,S);190(A,E,G,I,K,N,R,V,W); 192(A,I,L,M,P,R,T,V); 194(A,D,E,F,G,H,L,Q,T,V);196(A,Q,Y); 201(D,G,L,P); 202(I,M); 204(E,K,L,P,R); 208(E,M,P,R,W);210(A,F,L,Q); 211(C,E,G,P,S,V,W); 112(D,E,G,H,K,N,P,Q,R,S,T,V,W);213(E,H,L,M,P,Q,R,W); 220(A,C,D,G,L,P,R,S,T,W); 225(A,L,M,P,Q,S);226(C,E,F,G,P,R,W,Y); 227(A,C,G,P,R,S,V,W); 228(A,C,D,G,L,P,R,V,W);229(A,I,L,N,R,S,T); 230(A,G,H,L,P,Q,R,S,T,V,W);231(A,C,G,H,K,R,S,V,W,Y); 233(A,F,H,L,M,S,T); 236(C,L,P,S,V);237(F,G,H,L,M,N,R,V); 238(A,E,F,G,H,M,P,R,S,V,W); 239(F,G,S,Y); 240(A);241(M,V); 242(A,C,G,I,L,M,P,R,T,V,Y); 243(A,C,E,F,G,M);246(A,E,K,P,S,T,V); 247(A,E,L,P,T,Y); 248(A,G,P,V);252(A,D,E,G,N,Q,S,V); 255(C,E,M,Q,R);259(A,C,D,E,G,I,K,L,M,Q,R,T,V,W,Y); 262(A,D,E,K,M,R,T,V);264(C,G,I,L,M,P,R,S,T); 267(E,H,K,M,Q,W);269(A,D,E,G,H,K,L,M,N,Q,R,S,V,Y) of SEQ ID NO: 3.

The non-limiting list of composition components illustrated hereinafterare suitable for use in the compositions and methods herein may bedesirably incorporated in certain embodiments of the invention, e.g. toassist or enhance cleaning performance, for treatment of the substrateto be cleaned, or to modify the aesthetics of the composition as is thecase with perfumes, colorants, dyes or the like. The levels of any suchcomponents incorporated in any compositions are in addition to anymaterials previously recited for incorporation. The precise nature ofthese additional components, and levels of incorporation thereof, willdepend on the physical form of the composition and the nature of thecleaning operation for which it is to be used. Although componentsmentioned below are categorized by general header according to aparticular functionality, this is not to be construed as a limitation,as a component may comprise additional functionalities as will beappreciated by the skilled artisan.

Unless otherwise indicated the amounts in percentage is by weight of thecomposition (wt %). Suitable component materials include, but are notlimited to, surfactants, builders, chelating agents, dye transferinhibiting agents, dispersants, enzymes, and enzyme stabilizers,catalytic materials, bleach activators, hydrogen peroxide, sources ofhydrogen peroxide, preformed peracids, polymeric dispersing agents, claysoil removal/anti-redeposition agents, brighteners, suds suppressors,dyes, hueing dyes, perfumes, perfume delivery systems, structureelasticizing agents, fabric softeners, carriers, hydrotropes, processingaids, solvents and/or pigments. In addition to the disclosure below,suitable examples of such other components and levels of use are foundin U.S. Pat. No. 5,576,282, U.S. pat. No. 6,306,812, and U.S. Pat. No.6,326,348 hereby incorporated by reference.

Thus, in certain embodiments the invention do not contain one or more ofthe following adjuncts materials: surfactants, soaps, builders,chelating agents, dye transfer inhibiting agents, dispersants,additional enzymes, enzyme stabilizers, catalytic materials, bleachactivators, hydrogen peroxide, sources of hydrogen peroxide, preformedperacids, polymeric dispersing agents, clay soilremoval/anti-redeposition agents, brighteners, suds suppressors, dyes,perfumes, perfume delivery systems, structure elasticizing agents,fabric softeners, carriers, hydrotropes, processing aids, solventsand/or pigments. However, when one or more components are present, suchone or more components may be present as detailed below:

Surfactants—The compositions according to the present invention maycomprise a surfactant or surfactant system wherein the surfactant can beselected from nonionic surfactants, anionic surfactants, cationicsurfactants, ampholytic surfactants, zwitterionic surfactants,semi-polar nonionic surfactants and mixtures thereof. When present,surfactant is typically present at a level of from 0.1 to 60 wt %, from0,2 to 40 wt %, from 0,5 to 30 wt %, from 1 to 50 wt %, from 1 to 40 wt%, from 1 to 30 wt %, from 1 to 20 wt %, from 3 to 10 wt %, from 3 to 5wt %, from 5 to 40 wt %, from 5 to 30 wt %, from 5 to 15 wt %, from 3 to20 wt %, from 3 to 10 wt %, from 8 to 12 wt %, from 10 to 12 wt % orfrom 20 to 25 wt %.

Suitable anionic detersive surfactants include sulphate and sulphonatedetersive surfactants.

Suitable sulphonate detersive surfactants include alkyl benzenesulphonate, in one aspect, C₁₀₋₁₃ alkyl benzene sulphonate. Suitablealkyl benzene sulphonate (LAS) may be obtained, by sulphonatingcommercially available linear alkyl benzene (LAB); suitable LAB includeslow 2-phenyl LAB, such as Isochem® or Petrelab®, other suitable LABinclude high 2-phenyl LAB, such as Hyblene®. A suitable anionicdetersive surfactant is alkyl benzene sulphonate that is obtained byDETAL catalyzed process, although other synthesis routes, such as HF,may also be suitable. In one aspect a magnesium salt of LAS is used.

Suitable sulphate detersive surfactants include alkyl sulphate, in oneaspect, C₈₋₁₈ alkyl sulphate, or predominantly C₁₂ alkyl sulphate.

Another suitable sulphate detersive surfactant is alkyl alkoxylatedsulphate, in one aspect, alkyl ethoxylated sulphate, in one aspect, aC₈₋₁₈ alkyl alkoxylated sulphate, in another aspect,a C₈₋₁₈ alkylethoxylated sulphate, typically the alkyl alkoxylated sulphate has anaverage degree of alkoxylation of from 0.5 to 20, or from 0.5 to 10,typically the alkyl alkoxylated sulphate is a C₈-₁₈ alkyl ethoxylatedsulphate having an average degree of ethoxylation of from 0.5 to 10,from 0.5 to 7, from 0.5 to 5 or from 0.5 to 3.

The alkyl sulphate, alkyl alkoxylated sulphate and alkyl benzenesulphonates may be linear or branched, substituted or un-substituted.

The detersive surfactant may be a mid-chain branched detersivesurfactant, in one aspect, a mid-chain branched anionic detersivesurfactant, in one aspect, a mid-chain branched alkyl sulphate and/or amid-chain branched alkyl benzene sulphonate, e.g. a mid-chain branchedalkyl sulphate. In one aspect, the mid-chain branches are C₁₋₄ alkylgroups, typically methyl and/or ethyl groups.

Non-limiting examples of anionic surfactants include sulfates andsulfonates, in particular, linear alkylbenzenesulfonates (LAS), isomersof LAS, branched alkylbenzenesulfonates (BABS), phenylalkanesulfonates,alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates,alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates,alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcoholsulfates (FAS), primary alcohol sulfates (PAS), alcohol ethersulfates(AES or AEOS or FES, also known as alcohol ethoxysulfates or fattyalcohol ether sulfates), secondary alkanesulfonates (SAS), paraffinsulfonates (PS), ester sulfonates, sulfonated fatty acid glycerolesters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES)including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid,dodecenyl/tetradecenyl succinic acid (DTSA), fatty acid derivatives ofamino acids, diesters and monoesters of sulfo-succinic acid or soap, andcombinations thereof.

Suitable non-ionic detersive surfactants are selected from the groupconsisting of: C₈-C₁₈ alkyl ethoxylates, such as, NEODOL®; C₆-C₁₂ alkylphenol alkoxylates wherein the alkoxylate units may be ethyleneoxyunits, propyleneoxy units or a mixture thereof; C₁₂-C₁₈ alcohol andC₆-C₁₂ alkyl phenol condensates with ethylene oxide/propylene oxideblock polymers such as Pluronic®; C₁₄-C₂₂ mid-chain branched alcohols;C₁₄ ^(-C) ₂₂ mid-chain branched alkyl alkoxylates, typically having anaverage degree of alkoxylation of from 1 to 30; alkylpolysaccharides, inone aspect, alkylpolyglycosides; polyhydroxy fatty acid amides; ethercapped poly(oxyalkylated) alcohol surfactants; and mixtures thereof.

Suitable non-ionic detersive surfactants include alkyl polyglucosideand/or an alkyl alkoxylated alcohol.

In one aspect, non-ionic detersive surfactants include alkyl alkoxylatedalcohols, in one aspect C₈₋₁₈ alkyl alkoxylated alcohol, e.g. a C₈₋₁₈alkyl ethoxylated alcohol, the alkyl alkoxylated alcohol may have anaverage degree of alkoxylation of from 1 to 50, from 1 to 30, from 1 to20, or from 1 to 10. In one aspect, the alkyl alkoxylated alcohol may bea C₈₋₁₈ alkyl ethoxylated alcohol having an average degree ofethoxylation of from 1 to 10, from 1 to 7, more from 1 to 5 or from 3 to7. The alkyl alkoxylated alcohol can be linear or branched, andsubstituted or un-substituted. Suitable nonionic surfactants includeLutensol®.

Non-limiting examples of nonionic surfactants include alcoholethoxylates (AE or AEO), alcohol propoxylates, propoxylated fattyalcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylatedand/or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates(APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG),alkoxylated amines, fatty acid monoethanolamides (FAM), fatty aciddiethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM),propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fattyacid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides,GA, or fatty acid glucamides, FAGA), as well as products available underthe trade names SPAN and TWEEN, and combinations thereof.

Suitable cationic detersive surfactants include alkyl pyridiniumcompounds, alkyl quaternary ammonium compounds, alkyl quaternaryphosphonium compounds, alkyl ternary sulphonium compounds, and mixturesthereof.

Suitable cationic detersive surfactants are quaternary ammoniumcompounds having the general formula: (R)(R₁)(R₂)(R₃)N⁺X⁻, wherein, R isa linear or branched, substituted or unsubstituted C₆₋₁₈ alkyl oralkenyl moiety, R₁ and R₂ are independently selected from methyl orethyl moieties, R₃ is a hydroxyl, hydroxymethyl or a hydroxyethylmoiety, X is an anion which provides charge neutrality, suitable anionsinclude: halides, e.g. chloride; sulphate; and sulphonate. Suitablecationic detersive surfactants are mono-C₆₋₁₈ alkyl mono-hydroxyethyldi-methyl quaternary ammonium chlorides. Highly suitable cationicdetersive surfactants are mono-C₈₋₁₀ alkyl mono-hydroxyethyl di-methylquaternary ammonium chloride, mono-C₁₀₋₁₂ alkyl mono-hydroxyethyldi-methyl quaternary ammonium chloride and mono-C₁₀ alkylmono-hydroxyethyl di-methyl quaternary ammonium chloride.

Non-limiting examples of cationic surfactants includealkyldimethylethanolamine quat (ADMEAQ), cetyltrimethylammonium bromide(CTAB), dimethyldistearylammonium chloride (DSDMAC), andalkylbenzyldimethylammonium, alkyl quaternary ammonium compounds,alkoxylated quaternary ammonium (AQA) compounds, ester quats, andcombinations thereof.

Suitable amphoteric/zwitterionic surfactants include amine oxides andbetaines such as alkyldimethylbetaines, sulfobetaines, or combinationsthereof. Amine-neutralized anionic surfactants—Anionic surfactants ofthe present invention and adjunct anionic cosurfactants, may exist in anacid form, and said acid form may be neutralized to form a surfactantsalt which is desirable for use in the present detergent compositions.Typical agents for neutralization include the metal counterion base suchas hydroxides, eg, NaOH or KOH. Further preferred agents forneutralizing anionic surfactants of the present invention and adjunctanionic surfactants or cosurfactants in their acid forms includeammonia, amines, or alkanolamines. Alkanolamines are preferred. Suitablenon-limiting examples including monoethanolamine, diethanolamine,triethanolamine, and other linear or branched alkanolamines known in theart; e.g., highly preferred alkanolamines include 2-amino-1-propanol,1-aminopropanol, monoisopropanolamine, or 1-amino-3-propanol. Amineneutralization may be done to a full or partial extent, e.g. part of theanionic surfactant mix may be neutralized with sodium or potassium andpart of the anionic surfactant mix may be neutralized with amines oralkanolamines.

Non-limiting examples of semipolar surfactants include amine oxides (AO)such as alkyldimethylamineoxide

Surfactant systems comprising mixtures of one or more anionic and inaddition one or more nonionic surfactants optionally with an additionalsurfactant such as a cationic surfactant, may be preferred. Preferredweight ratios of anionic to nonionic surfactant are at least 2:1, or atleast 1:1 to 1:10.

Soap—The compositions herein may contain soap. Without being limited bytheory, it may be desirable to include soap as it acts in part as asurfactant and in part as a builder and may be useful for suppression offoam and may furthermore interact favorably with the various cationiccompounds of the composition to enhance softness on textile fabricstreaded with the inventive compositions. Any soap known in the art foruse in laundry detergents may be utilized. In one embodiment, thecompositions contain from 0 wt % to 20 wt %, from 0.5 wt % to 20 wt %,from 4 wt % to 10 wt %, or from 4 wt % to 7 wt % of soap.

Examples of soap useful herein include oleic acid soaps, palmitic acidsoaps, palm kernel fatty acid soaps, and mixtures thereof. Typical soapsare in the form of mixtures of fatty acid soaps having different chainlengths and degrees of substitution. One such mixture is topped palmkernel fatty acid.

In one embodiment, the soap is selected from free fatty acid. Suitablefatty acids are saturated and/or unsaturated and can be obtained fromnatural sources such a plant or animal esters (e.g., palm kernel oil,palm oil, coconut oil, babassu oil, safflower oil, tall oil, castor oil,tallow and fish oils, grease, and mixtures thereof), or syntheticallyprepared (e.g., via the oxidation of petroleum or by hydrogenation ofcarbon monoxide via the Fisher Tropsch process).

Examples of suitable saturated fatty acids for use in the compositionsof this invention include captic, lauric, myristic, palmitic, stearic,arachidic and behenic acid. Suitable unsaturated fatty acid speciesinclude: palmitoleic, oleic, linoleic, linolenic and ricinoleic acid.Examples of preferred fatty acids are saturated Cn fatty acid, saturatedCi₂-Ci₄ fatty acids, and saturated or unsaturated Cn to Ci₈ fatty acids,and mixtures thereof.

When present, the weight ratio of fabric softening cationic cosurfactantto fatty acid is preferably from about 1:3 to about 3: 1, morepreferably from about 1:1.5 to about 1.5:1, most preferably about 1:1.

Levels of soap and of nonsoap anionic surfactants herein are percentagesby weight of the detergent composition, specified on an acid form basis.However, as is commonly understood in the art, anionic surfactants andsoaps are in practice neutralized using sodium, potassium oralkanolammonium bases, such as sodium hydroxide or monoethanolamine.

Hydrotropes—The compositions of the present invention may comprise oneor more hydrotropes. A hydrotrope is a compound that solubiliseshydrophobic compounds in aqueous solutions (or oppositely, polarsubstances in a non-polar environment). Typically, hydrotropes have bothhydrophilic and a hydrophobic character (so-called amphiphilicproperties as known from surfactants); however the molecular structureof hydrotropes generally do not favor spontaneous self-aggregation, seee.g. review by Hodgdon and Kaler (2007), Current Opinion in Colloid &Interface Science 12: 121-128. Hydrotropes do not display a criticalconcentration above which self-aggregation occurs as found forsurfactants and lipids forming miceller, lamellar or other well definedmeso-phases. Instead, many hydrotropes show a continuous-typeaggregation process where the sizes of aggregates grow as concentrationincreases. However, many hydrotropes alter the phase behavior,stability, and colloidal properties of systems containing substances ofpolar and non-polar character, including mixtures of water, oil,surfactants, and polymers. Hydrotropes are classically used acrossindustries from pharma, personal care, food, to technical applications.Use of hydrotropes in detergent compositions allow for example moreconcentrated formulations of surfactants (as in the process ofcompacting liquid detergents by removing water) without inducingundesired phenomena such as phase separation or high viscosity.

The detergent may contain from 0 to 10 wt %, such as from 0 to 5 wt %,0.5 to 5 wt %, or from 3% to 5 wt %, of a hydrotrope. Any hydrotropeknown in the art for use in detergents may be utilized. Non-limitingexamples of hydrotropes include sodium benzenesulfonate, sodiump-toluene sulfonate (STS), sodium xylene sulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols andpolyglycolethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium ethylhexyl sulfate, and combinations thereof.

Builders—The compositions of the present invention may comprise one ormore builders, co-builders, builder systems or a mixture thereof. When abuilder is used, the cleaning composition will typically comprise from 0to 65 wt %, at least 1 wt %, from 2 to 60 wt % or from 5 to 10 wt %builder. In a dish wash cleaning composition, the level of builder istypically 40 to 65 wt % or 50 to 65 wt %. The composition may besubstantially free of builder; substantially free means “no deliberatelyadded” zeolite and/or phosphate. Typical zeolite builders includezeolite A, zeolite P and zeolite MAP. A typical phosphate builder issodium tri-polyphosphate.

The builder and/or co-builder may particularly be a chelating agent thatforms water-soluble complexes with Ca and Mg. Any builder and/orco-builder known in the art for use in detergents may be utilized.Non-limiting examples of builders include zeolites, diphosphates(pyrophosphates), triphosphates such as sodium triphosphate (STP orSTPP), carbonates such as sodium carbonate, soluble silicates such assodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst),ethanolamines such as 2-aminoethan-1-ol (MEA), iminodiethanol (DEA) and2,2′,2″-nitrilotriethanol (TEA), and carboxymethylinulin (CMI), andcombinations thereof.

The cleaning composition may include a co-builder alone, or incombination with a builder, e.g. a zeolite builder. Non-limitingexamples of co-builders include homopolymers of polyacrylates orcopolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylicacid/maleic acid) (PAA/PMA). Further non-limiting examples includecitrate, chelators such as aminocarboxylates, aminopolycarboxylates andphosphonates, and alkyl- or alkenylsuccinic acid. Additional specificexamples include 2,2′,2″-nitrilotriacetic acid (NTA),etheylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N′-disuccinicacid (EDDS), methylglycinediacetic acid (MGDA), glutamicacid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diylbis(phosphonicacid) (HEDP), ethylenediaminetetrakis(methylene)tetrakis(phosphonicacid) (EDTMPA), diethylenetriaminepentakis(methylene)pentakis(phosphonicacid) (DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), asparticacid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA),aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA),N-(2-sulfomethyl) aspartic acid (SMAS), N-(2-sulfoethyl) aspartic acid(SEAS), N-(2-sulfomethyl) glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA),α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA),isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid(PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilicacid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) andsulfomethyl-N,N-diacetic acid (SMDA),N-(hydroxyethyl)-ethylidenediaminetriacetate (HEDTA), diethanolglycine(DEG), Diethylenetriamine Penta (Methylene Phosphonic acid) (DTPMP),aminotris(methylenephosphonic acid) (ATMP), and combinations and saltsthereof. Further exemplary builders and/or co-builders are described in,e.g., WO09/102854, U.S. Pat. No. 5,977,053.

Chelating Agents and Crystal Growth Inhibitors—The compositions hereinmay contain a chelating agent and/or a crystal growth inhibitor.Suitable molecules include copper, iron and/or manganese chelatingagents and mixtures thereof. Suitable molecules include DTPA (Diethylenetriamine pentaacetic acid), HEDP (Hydroxyethane diphosphonic acid),DTPMP (Diethylene triamine penta(methylene phosphonic acid)),1,2-Dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate,ethylenediamine, diethylene triamine, ethylenediaminedisuccinic acid(EDDS), N-hydroxyethylethylenediaminetri-acetic acid (HEDTA),triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiaceticacid (HEIDA), dihydroxyethylglycine (DHEG),ethylenediaminetetrapropionic acid (EDTP), carboxymethyl inulin and2-Phosphonobutane 1,2,4-tricarboxylic acid (Bayhibit® AM) andderivatives thereof. Typically the composition may comprise from 0.005to 15 wt % or from 3.0 to 10 wt % chelating agent or crystal growthinhibitor.

Bleach Component—The bleach component suitable for incorporation in themethods and compositions of the invention comprise one or a mixture ofmore than one bleach component. Suitable bleach components includebleaching catalysts, photobleaches, bleach activators, hydrogenperoxide, sources of hydrogen peroxide, pre-formed peracids and mixturesthereof. In general, when a bleach component is used, the compositionsof the present invention may comprise from 0 to 30 wt %, from 0.00001 to90 wt %, 0.0001 to 50 wt %, from 0.001 to 25 wt % or from 1 to 20 wt %.Examples of suitable bleach components include:

(1) Pre-formed peracids: Suitable preformed peracids include, but arenot limited to, compounds selected from the group consisting ofpre-formed peroxyacids or salts thereof, typically either aperoxycarboxylic acid or salt thereof, or a peroxysulphonic acid or saltthereof. The pre-formed peroxyacid or salt thereof is preferably aperoxycarboxylic acid or salt thereof, typically having a chemicalstructure corresponding to the following chemical formula:

wherein: R¹⁴ is selected from alkyl, aralkyl, cycloalkyl, aryl orheterocyclic groups; the R¹⁴ group can be linear or branched,substituted or unsubstituted; and Y is any suitable counter-ion thatachieves electric charge neutrality, preferably Y is selected fromhydrogen, sodium or potassium. Preferably, R¹⁴ is a linear or branched,substituted or unsubstituted C₆₋₉ alkyl. Preferably, the peroxyacid orsalt thereof is selected from peroxyhexanoic acid, peroxyheptanoic acid,peroxyoctanoic acid, peroxynonanoic acid, peroxydecanoic acid, any saltthereof, or any combination thereof. Particularly preferred peroxyacidsare phthalimido-peroxy-alkanoic acids, in particular c-phthahlimidoperoxy hexanoic acid (PAP). Preferably, the peroxyacid or salt thereofhas a melting point in the range of from 30° C. to 60° C.

The pre-formed peroxyacid or salt thereof can also be a peroxysulphonicacid or salt thereof, typically having a chemical structurecorresponding to the following chemical formula:

wherein: R¹⁵ is selected from alkyl, aralkyl, cycloalkyl, aryl orheterocyclic groups; the R¹⁵ group can be linear or branched,substituted or unsubstituted; and Z is any suitable counter-ion thatachieves electric charge neutrality, preferably Z is selected fromhydrogen, sodium or potassium. Preferably R¹⁵ is a linear or branched,substituted or unsubstituted C₆₋₉ alkyl. Preferably such bleachcomponents may be present in the compositions of the invention in anamount from 0.01 to 50 wt % or from 0.1 to 20 wt %.

(2) Sources of hydrogen peroxide include e.g., inorganic perhydratesalts, including alkali metal salts such as sodium salts of perborate(usually mono- or tetra-hydrate), percarbonate, persulphate,perphosphate, persilicate salts and mixtures thereof. In one aspect ofthe invention the inorganic perhydrate salts such as those selected fromthe group consisting of sodium salts of perborate, percarbonate andmixtures thereof. When employed, inorganic perhydrate salts aretypically present in amounts of 0.05 to 40 wt % or 1 to 30 wt % of theoverall composition and are typically incorporated into suchcompositions as a crystalline solid that may be coated. Suitablecoatings include: inorganic salts such as alkali metal silicate,carbonate or borate salts or mixtures thereof, or organic materials suchas water-soluble or dispersible polymers, waxes, oils or fatty soaps.Preferably such bleach components may be present in the compositions ofthe invention in an amount of 0.01 to 50 wt % or 0.1 to 20 wt %.

(3) The term bleach activator is meant herein as a compound which reactswith hydrogen peroxide to form a peracid via perhydrolysis. The peracidthus formed constitutes the activated bleach. Suitable bleach activatorsto be used herein include those belonging to the class of esters,amides, imides or anhydrides. Suitable bleach activators are thosehaving R—(C═O)-L wherein R is an alkyl group, optionally branched,having, when the bleach activator is hydrophobic, from 6 to 14 carbonatoms, or from 8 to 12 carbon atoms and, when the bleach activator ishydrophilic, less than 6 carbon atoms or less than 4 carbon atoms; and Lis leaving group. Examples of suitable leaving groups are benzoic acidand derivatives thereof—especially benzene sulphonate. Suitable bleachactivators include dodecanoyl oxybenzene sulphonate, decanoyl oxybenzenesulphonate, decanoyl oxybenzoic acid or salts thereof, 3,5,5-trimethylhexanoyloxybenzene sulphonate, tetraacetyl ethylene diamine (TAED),sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS),4-(dodecanoyloxy)benzene-1-sulfonate (LOBS),4-(decanoyloxy)benzene-1-sulfonate, 4-(decanoyloxy)benzoate (DOBS orDOBA), 4-(nonanoyloxy)benzene-1-sulfonate (NOBS), and/or those disclosedin WO98/17767. A family of bleach activators is disclosed in EP624154and particularly preferred in that family is acetyl triethyl citrate(ATC). ATC or a short chain triglyceride like triacetin has theadvantage that it is environmentally friendly. Furthermore acetyltriethyl citrate and triacetin have good hydrolytical stability in theproduct upon storage and are efficient bleach activators. Finally ATC ismultifunctional, as the citrate released in the perhydrolysis reactionmay function as a builder. Alternatively, the bleaching system maycomprise peroxyacids of, for example, the amide, imide, or sulfone type.The bleaching system may also comprise peracids such as6-(phthalimido)peroxyhexanoic acid (PAP). Suitable bleach activators arealso disclosed in WO98/17767. While any suitable bleach activator may beemployed, in one aspect of the invention the subject cleaningcomposition may comprise NOBS, TAED or mixtures thereof. When present,the peracid and/or bleach activator is generally present in thecomposition in an amount of 0.1 to 60 wt %, 0.5 to 40 wt % or 0.6 to 10wt % based on the fabric and home care composition. One or morehydrophobic peracids or precursors thereof may be used in combinationwith one or more hydrophilic peracid or precursor thereof. Preferablysuch bleach components may be present in the compositions of theinvention in an amount of 0.01 to 50 wt %, or 0.1 to 20 wt %.

The amounts of hydrogen peroxide source and peracid or bleach activatormay be selected such that the molar ratio of available oxygen (from theperoxide source) to peracid is from 1:1 to 35:1, or even 2:1 to 10:1.

(4) Diacyl peroxides—preferred diacyl peroxide bleaching species includethose selected from diacyl peroxides of the general formula:R¹—C(O)—OO—(O)C—R², in which R¹ represents a C₆-C₁₈ alkyl, preferablyC₆-C₁₂ alkyl group containing a linear chain of at least 5 carbon atomsand optionally containing one or more substituents (e.g. —N⁺(CH₃)₃,—COOH or —CN) and/or one or more interrupting moieties (e.g. —CONH— or—CH═CH—) interpolated between adjacent carbon atoms of the alkylradical, and R² represents an aliphatic group compatible with a peroxidemoiety, such that R¹ and R² together contain a total of 8 to 30 carbonatoms. In one preferred aspect R¹ and R² are linear unsubstituted C₆-C₁₂alkyl chains. Most preferably R¹ and R² are identical. Diacyl peroxides,in which both R¹ and R² are C₆-C₁₂ alkyl groups, are particularlypreferred. Preferably, at least one of, most preferably only one of, theR groups (R₁ or R₂), does not contain branching or pendant rings in thealpha position, or preferably neither in the alpha nor beta positions ormost preferably in none of the alpha or beta or gamma positions. In onefurther preferred embodiment the DAP may be asymmetric, such thatpreferably the hydrolysis of R1 acyl group is rapid to generate peracid,but the hydrolysis of R2 acyl group is slow.

The tetraacyl peroxide bleaching species is preferably selected fromtetraacyl peroxides of the general formula:R³—C(O)—OO—C(O)—(CH₂)n-C(O)—OO—C(O)—R³, in which R³ represents a C₁-C₉alkyl, or C₃-C₇, group and n represents an integer from 2 to 12, or 4 to10 inclusive.

Preferably, the diacyl and/or tetraacyl peroxide bleaching species ispresent in an amount sufficient to provide at least 0.5 ppm, at least 10ppm, or at least 50 ppm by weight of the wash liquor. In a preferredembodiment, the bleaching species is present in an amount sufficient toprovide from 0.5 to 300 ppm, from 30 to 150 ppm by weight of the washliquor. Preferably the bleach component comprises a bleach catalyst (5and 6).

(5) Preferred are organic (non-metal) bleach catalysts include bleachcatalyst capable of accepting an oxygen atom from a peroxyacid and/orsalt thereof, and transferring the oxygen atom to an oxidizeablesubstrate. Suitable bleach catalysts include, but are not limited to:iminium cations and polyions; iminium zwitterions; modified amines;modified amine oxides; N-sulphonyl imines; N-phosphonyl imines; N-acylimines; thiadiazole dioxides; perfluoroimines; cyclic sugar ketones andmixtures thereof.

Suitable iminium cations and polyions include, but are not limited to,N-methyl-3,4-dihydroisoquinolinium tetrafluoroborate, prepared asdescribed in Tetrahedron (1992), 49(2), 423-38 (e.g. compound 4, p.433);N-methyl-3,4-dihydroisoquinolinium p-toluene sulphonate, prepared asdescribed in US5360569 (e.g. Column 11, Example 1); andN-octyl-3,4-dihydroisoquinolinium p-toluene sulphonate, prepared asdescribed in U.S. Pat. No. 5,360,568 (e.g. Column 10, Ex. 3).

Suitable iminium zwitterions include, but are not limited to,N-(3-sulfopropyl)-3,4-dihydroisoquinolinium, inner salt, prepared asdescribed in U.S. Pat. No. 5,576,282 (e.g. Column 31, Ex. II);N-[2-(sulphooxy)dodecyl]-3,4-dihydroisoquinolinium, inner salt, preparedas described in U.S. Pat. No. 5,817,614 (e.g. Column 32, Ex. V);2[3-[(2-ethylhexyl)oxy]-2-(sulphooxy)propyl]-3,4-dihydroisoquinolinium,inner salt, prepared as described in WO05/047264 (e.g. p.18, Ex. 8), and2-[3-[(2-butyloctyl)oxy]-2-(sulphooxy)propyl]-3,4-dihydroisoquinolinium,inner salt.

Suitable modified amine oxygen transfer catalysts include, but are notlimited to, 1,2,3,4-tetrahydro-2-methyl-1-isoquinolinol, which can bemade according to the procedures described in Tetrahedron Letters(1987), 28(48), 6061-6064. Suitable modified amine oxide oxygen transfercatalysts include, but are not limited to, sodium1-hydroxy-N-oxy-N-[2-(sulphooxy)decyl]-1,2,3,4-tetrahydroisoquinoline.

Suitable N-sulphonyl imine oxygen transfer catalysts include, but arenot limited to, 3-methyl-1,2-benzisothiazole 1,1-dioxide, preparedaccording to the procedure described in the Journal of Organic Chemistry(1990), 55(4), 1254-61.

Suitable N-phosphonyl imine oxygen transfer catalysts include, but arenot limited to,[R-(E)]-N-[(2-chloro-5-nitrophenyl)methylene]-P-phenyl-P-(2,4,6-trimethylphenyl)-phosphinicamide, which can be made according to the procedures described in theJournal of the Chemical Society, Chemical Communications (1994), (22),2569-70.

Suitable N-acyl imine oxygen transfer catalysts include, but are notlimited to, [N(E)]-N-(phenylmethylene)acetamide, which can be madeaccording to the procedures described in Polish Journal of Chemistry(2003), 77(5), 577-590.

Suitable thiadiazole dioxide oxygen transfer catalysts include but arenot limited to, 3-methyl-4-phenyl-1,2,5-thiadiazole 1,1-dioxide, whichcan be made according to the procedures described in U.S. Pat. No.5,753,599 (Column 9, Ex. 2).

Suitable perfluoroimine oxygen transfer catalysts include, but are notlimited to,(Z)-2,2,3,3,4,4,4-heptafluoro-N-(nonafluorobutyl)butanimidoyl fluoride,which can be made according to the procedures described in TetrahedronLetters (1994), 35(34), 6329-30.

Suitable cyclic sugar ketone oxygen transfer catalysts include, but arenot limited to,1,2:4,5-di-O-isopropylidene-D-erythro-2,3-hexodiuro-2,6-pyranose asprepared in U.S. Pat. No. 6,649,085 (Column 12, Ex. 1).

Preferably, the bleach catalyst comprises an iminium and/or carbonylfunctional group and is typically capable of forming an oxaziridiniumand/or dioxirane functional group upon acceptance of an oxygen atom,especially upon acceptance of an oxygen atom from a peroxyacid and/orsalt thereof. Preferably, the bleach catalyst comprises an oxaziridiniumfunctional group and/or is capable of forming an oxaziridiniumfunctional group upon acceptance of an oxygen atom, especially uponacceptance of an oxygen atom from a peroxyacid and/or salt thereof.Preferably, the bleach catalyst comprises a cyclic iminium functionalgroup, preferably wherein the cyclic moiety has a ring size of from fiveto eight atoms (including the nitrogen atom), preferably six atoms.Preferably, the bleach catalyst comprises an aryliminium functionalgroup, preferably a bi-cyclic aryliminium functional group, preferably a3,4-dihydroisoquinolinium functional group. Typically, the iminefunctional group is a quaternary imine functional group and is typicallycapable of forming a quaternary oxaziridinium functional group uponacceptance of an oxygen atom, especially upon acceptance of an oxygenatom from a peroxyacid and/or salt thereof. In another aspect, thedetergent composition comprises a bleach component having a log P_(o/w)no greater than 0, no greater than −0.5, no greater than −1.0, nogreater than −1.5, no greater than −2.0, no greater than −2.5, nogreater than −3.0, or no greater than −3.5. The method for determininglog P_(o/w) is described in more detail below.

Typically, the bleach ingredient is capable of generating a bleachingspecies having a X_(SO) of from 0.01 to 0.30, from 0.05 to 0.25, or from0.10 to 0.20. The method for determining X_(SO) is described in moredetail below. For example, bleaching ingredients having anisoquinolinium structure are capable of generating a bleaching speciesthat has an oxaziridinium structure. In this example, the X_(SO) is thatof the oxaziridinium bleaching species.

Preferably, the bleach catalyst has a chemical structure correspondingto the following chemical formula:

wherein: n and m are independently from 0 to 4, preferably n and m areboth 0; each R¹ is independently selected from a substituted orunsubstituted radical selected from the group consisting of hydrogen,alkyl, cycloalkyl, aryl, fused aryl, heterocyclic ring, fusedheterocyclic ring, nitro, halo, cyano, sulphonato, alkoxy, keto,carboxylic, and carboalkoxy radicals; and any two vicinal R¹substituents may combine to form a fused aryl, fused carbocyclic orfused heterocyclic ring; each R² is independently selected from asubstituted or unsubstituted radical independently selected from thegroup consisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkaryl, aryl,aralkyl, alkylenes, heterocyclic ring, alkoxys, arylcarbonyl groups,carboxyalkyl groups and amide groups; any R² may be joined together withany other of R² to form part of a common ring; any geminal R² maycombine to form a carbonyl; and any two R² may combine to form asubstituted or unsubstituted fused unsaturated moiety; R³ is a C₁ to C₂₀substituted or unsubstituted alkyl; R⁴ is hydrogen or the moietyQ_(t)-A, wherein: Q is a branched or unbranched alkylene, t=0 or 1 and Ais an anionic group selected from the group consisting of OSO₃ ⁻, SO₃ ⁻,CO₂ ⁻, OCO₂ ⁻; OPO₃ ²⁻, OPO₃H⁻ and OPO₂ ⁻; R⁵ is hydrogen or the moiety−CR¹¹R¹²—Y_(b)Y_(c)-[(CR⁹R¹⁰)_(y)—O]_(k)—R⁸, wherein: each Y isindependently selected from the group consisting of O, S, N—H, or N—R⁸;and each R⁸ is independently selected from the group consisting ofalkyl, aryl and heteroaryl, said moieties being substituted orunsubstituted, and whether substituted or unsubsituted said moietieshaving less than 21 carbons; each G is independently selected from thegroup consisting of CO, SO₂, SO, PO and PO₂; R⁹ and R¹⁰ areindependently selected from the group consisting of H and C₁-C₄ alkyl;R¹¹ and R¹² are independently selected from the group consisting of Hand alkyl, or when taken together may join to form a carbonyl; b=0 or 1;c can=0 or 1, but c must=0 if b=0; y is an integer from 1 to 6; k is aninteger from 0 to 20; R⁶ is H, or an alkyl, aryl or heteroaryl moiety;said moieties being substituted or unsubstituted; and X, if present, isa suitable charge balancing counterion, preferably X is present when R⁴is hydrogen, suitable X, include but are not limited to: chloride,bromide, sulphate, methosulphate, sulphonate, p-toluenesulphonate,borontetraflouride and phosphate.

In one embodiment of the present invention, the bleach catalyst has astructure corresponding to general formula below:

wherein R¹³ is a branched alkyl group containing from three to 24 carbonatoms (including the branching carbon atoms) or a linear alkyl groupcontaining from one to 24 carbon atoms; preferably R¹³ is a branchedalkyl group containing from eight to 18 carbon atoms or linear alkylgroup containing from eight to eighteen carbon atoms; preferably R¹³ isselected from the group consisting of 2-propylheptyl, 2-butyloctyl,2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl,n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl and iso-pentadecyl;preferably R¹³ is selected from the group consisting of 2-butyloctyl,2-pentylnonyl, 2-hexyldecyl, iso-tridecyl and iso-pentadecyl.

Preferably the bleach component comprises a source of peracid inaddition to bleach catalyst, particularly organic bleach catalyst. Thesource of peracid may be selected from (a) pre-formed peracid; (b)percarbonate, perborate or persulfate salt (hydrogen peroxide source)preferably in combination with a bleach activator; and (c) perhydrolaseenzyme and an ester for forming peracid in situ in the presence of waterin a textile or hard surface treatment step. When present, the peracidand/or bleach activator is generally present in the composition in anamount of from 0.1 to 60 wt %, from 0.5 to 40 wt % or from 0.6 to 10 wt% based on the composition. One or more hydrophobic peracids orprecursors thereof may be used in combination with one or morehydrophilic peracid or precursor thereof.

The amounts of hydrogen peroxide source and peracid or bleach activatormay be selected such that the molar ratio of available oxygen (from theperoxide source) to peracid is from 1:1 to 35:1, or 2:1 to 10:1.

(6) Metal-containing Bleach Catalysts—The bleach component may beprovided by a catalytic metal complex. One type of metal-containingbleach catalyst is a catalyst system comprising a transition metalcation of defined bleach catalytic activity, such as copper, iron,titanium, ruthenium, tungsten, molybdenum, or manganese cations, anauxiliary metal cation having little or no bleach catalytic activity,such as zinc or aluminum cations, and a sequestrate having definedstability constants for the catalytic and auxiliary metal cations,particularly ethylenediaminetetraacetic acid,ethylenediaminetetra(methylenephosphonic acid) and water-soluble saltsthereof. Such catalysts are disclosed in U.S. Pat. No. 4,430,243.Preferred catalysts are described in WO09/839406, U.S. Pat. No.6,218,351 and WO00/012667. Particularly preferred are transition metalcatalyst or ligands therefore that are cross-bridged polydentate N-donorligands.

If desired, the compositions herein can be catalyzed by means of amanganese compound. Such compounds and levels of use are well known inthe art and include, e.g., the manganese-based catalysts disclosed inU.S. Pat. No. 5,576,282.

Cobalt bleach catalysts useful herein are known, and are described e.g.in U.S. Pat. No. 5,597,936; U.S. Pat. No. 5,595,967. Such cobaltcatalysts are readily prepared by known procedures, such as taught e.g.in U.S. Pat. No. 5,597,936 and U.S. Pat. No. 5,595,967.

Compositions herein may also suitably include a transition metal complexof ligands such as bispidones (U.S. Pat. No. 7,501,389) and/ormacropolycyclic rigid ligands—abbreviated as “MRLs”. As a practicalmatter, and not by way of limitation, the compositions and processesherein can be adjusted to provide on the order of at least one part perhundred million of the active MRL species in the aqueous washing medium,and will typically provide from 0.005 to 25 ppm, from 0.05 to 10 ppm, orfrom 0.1 to 5 ppm, of the MRL in the wash liquor.

Suitable transition-metals in the instant transition-metal bleachcatalyst include e.g. manganese, iron and chromium. Suitable MRLsinclude 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane. Suitabletransition metal MRLs are readily prepared by known procedures, such astaught e.g. in U.S. Pat. No. 6,225,464 and WO00/32601.

(7) Photobleaches—suitable photobleaches include e.g. sulfonated zincphthalocyanine sulfonated aluminium phthalocyanines, xanthene dyes andmixtures thereof. Preferred bleach components for use in the presentcompositions of the invention comprise a hydrogen peroxide source,bleach activator and/or organic peroxyacid, optionally generated in situby the reaction of a hydrogen peroxide source and bleach activator, incombination with a bleach catalyst. Preferred bleach components comprisebleach catalysts, preferably organic bleach catalysts, as describedabove.

Particularly preferred bleach components are the bleach catalysts inparticular the organic bleach catalysts.

Exemplary bleaching systems are also described, e.g. in WO2007/087258,WO2007/087244, WO2007/087259 and WO2007/087242.

Fabric Hueing Agents—The composition may comprise a fabric hueing agent.Suitable fabric hueing agents include dyes, dye-clay conjugates, andpigments. Suitable dyes include small molecule dyes and polymeric dyes.Suitable small molecule dyes include small molecule dyes selected fromthe group consisting of dyes falling into the Color Index (C.I.)classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue,Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, ormixtures thereof.

In another aspect, suitable small molecule dyes include small moleculedyes selected from the group consisting of Color Index (Society of Dyersand Colorists, Bradford, UK) numbers Direct Violet 9, Direct Violet 35,Direct Violet 48, Direct Violet 51, Direct Violet 66, Direct Violet 99,Direct Blue 1, Direct Blue 71, Direct Blue 80, Direct Blue 279, Acid Red17, Acid Red 73, Acid Red 88, Acid Red 150, Acid Violet 15, Acid Violet17, Acid Violet 24, Acid Violet 43, Acid Red 52, Acid Violet 49, AcidViolet 50, Acid Blue 15, Acid Blue 17, Acid Blue 25, Acid Blue 29, AcidBlue 40, Acid Blue 45, Acid Blue 75, Acid Blue 80, Acid Blue 83, AcidBlue 90 and Acid Blue 113, Acid Black 1, Basic Violet 1, Basic Violet 3,Basic Violet 4, Basic Violet 10, Basic Violet 35, Basic Blue 3, BasicBlue 16, Basic Blue 22, Basic Blue 47, Basic Blue 66, Basic Blue 75,Basic Blue 159 and mixtures thereof. In another aspect, suitable smallmolecule dyes include small molecule dyes selected from the groupconsisting of Color Index (Society of Dyers and Colorists, Bradford, UK)numbers Acid Violet 17, Acid Violet 43, Acid Red 52, Acid Red 73, AcidRed 88, Acid Red 150, Acid Blue 25, Acid Blue 29, Acid Blue 45, AcidBlue 113, Acid Black 1, Direct Blue 1, Direct Blue 71, Direct Violet 51and mixtures thereof. In another aspect, suitable small molecule dyesinclude small molecule dyes selected from the group consisting of ColorIndex (Society of Dyers and Colorists, Bradford, UK) numbers Acid Violet17, Direct Blue 71, Direct Violet 51, Direct Blue 1, Acid Red 88, AcidRed 150, Acid Blue 29, Acid Blue 113 or mixtures thereof.

Suitable polymeric dyes include polymeric dyes selected from the groupconsisting of polymers containing conjugated chromogens (dye-polymerconjugates) and polymers with chromogens co-polymerized into thebackbone of the polymer and mixtures thereof.

In another aspect, suitable polymeric dyes include polymeric dyesselected from the group consisting of fabric-substantive colorants soldunder the name of Liquitint® (Milliken), dye-polymer conjugates formedfrom at least one reactive dye and a polymer selected from the groupconsisting of polymers comprising a moiety selected from the groupconsisting of a hydroxyl moiety, a primary amine moiety, a secondaryamine moiety, a thiol moiety and mixtures thereof. In still anotheraspect, suitable polymeric dyes include polymeric dyes selected from thegroup consisting of Liquitint® Violet CT, carboxymethyl cellulose (CMC)conjugated with a reactive blue, reactive violet or reactive red dyesuch as CMC conjugated with C.I. Reactive Blue 19, sold by Megazyme,Wicklow, Ireland under the product name AZO-CM-CELLULOSE, product codeS-ACMC, alkoxylated triphenyl-methane polymeric colorants, alkoxylatedthiophene polymeric colorants, and mixtures thereof.

Preferred hueing dyes include the whitening agents found in WO08/87497.These whitening agents may be characterized by the following structure(I):

wherein R₁ and R₂ can independently be selected from:

-   a) [(CH₂CR′HO)_(x)(CH₂CR″HO)_(y)H]    wherein R′ is selected from the group consisting of H, CH₃,    CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected    from the group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures    thereof; wherein x+y≦5; wherein y≧1; and wherein z=0 to 5;-   b) R₁=alkyl, aryl or aryl alkyl and    R₂=[(CH₂CR′HO)_(x)(CH₂CR″HO)_(y)H]    wherein R′ is selected from the group consisting of H, CH₃,    CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected    from the group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures    thereof; wherein x+y≦10; wherein y≧1; and wherein z=0 to 5;-   c) R₁=[CH₂CH₂(OR₃)CH₂OR₄] and R₂=[CH₂CH₂(OR₃)CH₂OR₄]    wherein R₃ is selected from the group consisting of H,    (CH₂CH₂O)_(z)H, and mixtures thereof; and wherein z =0 to 10;    wherein R₄ is selected from the group consisting of (C₁-C₁₆)alkyl ,    aryl groups, and mixtures thereof; and-   d) wherein R1 and R2 can independently be selected from the amino    addition product of styrene oxide, glycidyl methyl ether, isobutyl    glycidyl ether, isopropylglycidyl ether, t-butyl glycidyl ether,    2-ethylhexylgycidyl ether, and glycidylhexadecyl ether, followed by    the addition of from 1 to 10 alkylene oxide units.

A preferred whitening agent of the present invention may becharacterized by the following structure (II):

wherein R′ is selected from the group consisting of H, CH₃,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected fromthe group consisting of H, CH₂O(CH₂CH₂O)₂H, and mixtures thereof;wherein x+y≦5; wherein y≧1; and wherein z=0 to 5.

A further preferred whitening agent of the present invention may becharacterized by the following structure (III):

typically comprising a mixture having a total of 5 EO groups. Suitablepreferred molecules are those in Structure I having the followingpendant groups in “part a” above.

TABLE A R1 R2 R′ R″ x y R′ R″ x y A H H 3 1 H H 0 1 B H H 2 1 H H 1 1 c= b H H 1 1 H H 2 1 d = a H H 0 1 H H 3 1

Further whitening agents of use include those described in US2008/34511(Unilever). A preferred agent is “Violet 13”.

Suitable dye clay conjugates include dye clay conjugates selected fromthe group comprising at least one cationic/basic dye and a smectiteclay, and mixtures thereof. In another aspect, suitable dye clayconjugates include dye clay conjugates selected from the groupconsisting of one cationic/basic dye selected from the group consistingof C.I. Basic Yellow 1 through 108, C.I. Basic Orange 1 through 69, C.I.Basic Red 1 through 118, C.I. Basic Violet 1 through 51, C.I. Basic Blue1 through 164, C.I. Basic Green 1 through 14, C.I. Basic Brown 1 through23, CI Basic Black 1 through 11, and a clay selected from the groupconsisting of Montmorillonite clay, Hectorite clay, Saponite clay andmixtures thereof. In still another aspect, suitable dye clay conjugatesinclude dye clay conjugates selected from the group consisting of:Montmorillonite Basic Blue B7 C.I. 42595 conjugate, MontmorilloniteBasic Blue B9 C.I. 52015 conjugate, Montmorillonite Basic Violet V3 C.I.42555 conjugate, Montmorillonite Basic Green G1 C.I. 42040 conjugate,Montmorillonite Basic Red R1 C.I. 45160 conjugate, Montmorillonite C.I.Basic Black 2 conjugate, Hectorite Basic Blue B7 C.I. 42595 conjugate,Hectorite Basic Blue B9 C.I. 52015 conjugate, Hectorite Basic Violet V3C.I. 42555 conjugate, Hectorite Basic Green G1 C.I. 42040 conjugate,Hectorite Basic Red R1 C.I. 45160 conjugate, Hectorite C.I. Basic Black2 conjugate, Saponite Basic Blue B7 C.I. 42595 conjugate, Saponite BasicBlue B9 C.I. 52015 conjugate, Saponite Basic Violet V3 C.I. 42555conjugate, Saponite Basic Green G1 C.I. 42040 conjugate, Saponite BasicRed R1 C.I. 45160 conjugate, Saponite C.I. Basic Black 2 conjugate andmixtures thereof.

Suitable pigments include pigments selected from the group consisting offlavanthrone, indanthrone, chlorinated indanthrone containing from 1 to4 chlorine atoms, pyranthrone, dichloropyranthrone,monobromodichloropyranthrone, dibromodichloropyranthrone,tetrabromopyranthrone, perylene-3,4,9,10-tetracarboxylic acid diimide,wherein the imide groups may be unsubstituted or substituted byC1-C3-alkyl or a phenyl or heterocyclic radical, and wherein the phenyland heterocyclic radicals may additionally carry substituents which donot confer solubility in water, anthrapyrimidinecarboxylic acid amides,violanthrone, isoviolanthrone, dioxazine pigments, copper phthalocyaninewhich may contain up to 2 chlorine atoms per molecule, polychloro-copperphthalocyanine or polybromochloro-copper phthalocyanine containing up to14 bromine atoms per molecule and mixtures thereof.

In another aspect, suitable pigments include pigments selected from thegroup consisting of Ultramarine Blue (CA. Pigment Blue 29), UltramarineViolet (CA. Pigment Violet 15) and mixtures thereof.

The aforementioned fabric hueing agents can be used in combination (anymixture of fabric hueing agents can be used). Suitable hueing agents aredescribed in more detail in U.S. Pat. No. 7,208,459. Preferred levels ofdye in compositions of the invention are 0.00001 to 0.5 wt %, or 0.0001to 0.25 wt %. The concentration of dyes preferred in water for thetreatment and/or cleaning step is from 1 ppb to 5 ppm, 10 ppb to 5 ppmor 20 ppb to 5 ppm. In preferred compositions, the concentration ofsurfactant will be from 0.2 to 3 g/l.

Encapsulates—The composition may comprise an encapsulate. In one aspect,an encapsulate comprising a core, a shell having an inner and outersurface, said shell encapsulating said core.

In one aspect of said encapsulate, said core may comprise a materialselected from the group consisting of perfumes; brighteners; dyes;insect repellants; silicones; waxes; flavors; vitamins; fabric softeningagents; skin care agents in one aspect, paraffins; enzymes;anti-bacterial agents; bleaches; sensates; and mixtures thereof; andsaid shell may comprise a material selected from the group consisting ofpolyethylenes; polyamides; polyvinylalcohols, optionally containingother co-monomers; polystyrenes; polyisoprenes; polycarbonates;polyesters; polyacrylates; aminoplasts, in one aspect said aminoplastmay comprise a polyureas, polyurethane, and/or polyureaurethane, in oneaspect said polyurea may comprise polyoxymethyleneurea and/or melamineformaldehyde; polyolefins; polysaccharides, in one aspect saidpolysaccharide may comprise alginate and/or chitosan; gelatin; shellac;epoxy resins; vinyl polymers; water insoluble inorganics; silicone; andmixtures thereof.

In one aspect of said encapsulate, said core may comprise perfume.

In one aspect of said encapsulate, said shell may comprise melamineformaldehyde and/or cross linked melamine formaldehyde.

In a one aspect, suitable encapsulates may comprise a core material anda shell, said shell at least partially surrounding said core material,is disclosed. 85% or 90% of said encapsulates may have a fracturestrength of from 0.2 to 10 MPa, from 0.4 to 5 MPa, from 0.6 to 3.5 MPa,or from 0.7 to 3 MPa; and a benefit agent leakage of from 0 to 30%, from0 to 20%, or from 0 to 5%.

In one aspect, 85% or 90% of said encapsulates may have a particle sizefrom 1 to 80 microns, from 5 to 60 microns, from 10 to 50 microns, orfrom 15 to 40 microns.

In one aspect, 85% or 90% of said encapsulates may have a particle wallthickness from 30 to 250 nm, from 80 to 180 nm, or from 100 to 160 nm.

In one aspect, said encapsulates' core material may comprise a materialselected from the group consisting of a perfume raw material and/oroptionally a material selected from the group consisting of vegetableoil, including neat and/or blended vegetable oils including castor oil,coconut oil, cottonseed oil, grape oil, rapeseed, soybean oil, corn oil,palm oil, linseed oil, safflower oil, olive oil, peanut oil, coconutoil, palm kernel oil, castor oil, lemon oil and mixtures thereof; estersof vegetable oils, esters, including dibutyl adipate, dibutyl phthalate,butyl benzyl adipate, benzyl octyl adipate, tricresyl phosphate,trioctyl phosphate and mixtures thereof; straight or branched chainhydrocarbons, including those straight or branched chain hydrocarbonshaving a boiling point of greater than about 80° C.; partiallyhydrogenated terphenyls, dialkyl phthalates, alkyl biphenyls, includingmonoisopropylbiphenyl, alkylated naphthalene, includingdipropylnaphthalene, petroleum spirits, including kerosene, mineral oiland mixtures thereof; aromatic solvents, including benzene, toluene andmixtures thereof; silicone oils; and mixtures thereof.

In one aspect, said encapsulates' wall material may comprise a suitableresin including the reaction product of an aldehyde and an amine,suitable aldehydes include, formaldehyde. Suitable amines includemelamine, urea, benzoguanamine, glycoluril, and mixtures thereof.Suitable melamines include methylol melamine, methylated methylolmelamine, imino melamine and mixtures thereof. Suitable ureas includedimethylol urea, methylated dimethylol urea, urea-resorcinol, andmixtures thereof.

In one aspect, suitable formaldehyde scavengers may be employed with theencapsulates e.g. in a capsule slurry and/or added to a compositionbefore, during or after the encapsulates are added to such composition.Suitable capsules may be made by the following teaching ofUS2008/0305982; and/or US2009/0247449.

In a preferred aspect the composition can also comprise a depositionaid, preferably consisting of the group comprising cationic or nonionicpolymers. Suitable polymers include cationic starches, cationichydroxyethylcellulose, polyvinylformaldehyde, locust bean gum, mannans,xyloglucans, tamarind gum, polyethyleneterephthalate and polymerscontaining dimethylaminoethyl methacrylate, optionally with one ormonomers selected from the group comprising acrylic acid and acrylamide.

Perfumes—In one aspect the composition comprises a perfume thatcomprises one or more perfume raw materials selected from the groupconsisting of 1,1′-oxybis-2-propanol; 1,4-cyclohexanedicarboxylic acid,diethyl ester; (ethoxymethoxy)cyclododecane; 1,3-nonanediol,monoacetate; (3-methylbutoxy)acetic acid, 2-propenyl ester; beta-methylcyclododecaneethanol;2-methyl-3-[(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)oxy]-1-propanol;oxacyclohexadecan-2-one; alpha-methyl-benzenemethanol acetate;trans-3-ethoxy-1,1,5-trimethylcyclohexane;4-(1,1-dimethylethyl)cyclohexanol acetate;dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan; beta-methylbenzenepropanal; beta-methyl-3-(1-methylethyl)benzenepropanal;4-phenyl-2-butanone; 2-methylbutanoic acid, ethyl ester; benzaldehyde;2-methylbutanoic acid, 1-methylethyl ester;dihydro-5-pentyl-2(3H)furanone;(2E)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)-2-buten-1-one; dodecanal;undecanal; 2-ethyl-alpha, alpha-dimethylbenzenepropanal; decanal; alpha,alpha-dimethylbenzeneethanol acetate; 2-(phenylmethylene)octanal;2-[[3-[4-(1,1-dimethylethyl)phenyl]-2-methylpropylidene]amino]benzoicacid, methyl ester; 1-(2,6,6-trimethyl-3-cyclohexen-1-yl)-2-buten-1-one;2-pentylcyclopentanone; 3-oxo-2-pentyl cyclopentaneacetic acid, methylester; 4-hydroxy-3-methoxybenzaldehyde; 3-ethoxy-4-hydroxybenzaldehyde;2-heptylcyclopentanone; 1-(4-methylphenyl)ethanone;(3E)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one;(3E)-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one;benzeneethanol; 2H-1-benzopyran-2-one; 4-methoxybenzaldehyde;10-undecenal; propanoic acid, phenylmethyl ester;beta-methylbenzenepentanol; 1,1-diethoxy-3,7-dimethyl-2,6-octadiene;alpha, alpha-dimethylbenzeneethanol;(2E)-1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2-buten-1-one; acetic acid,phenylmethyl ester; cyclohexanepropanoic acid, 2-propenyl ester;hexanoic acid, 2-propenyl ester; 1,2-dimethoxy-4-(2-propenyl)benzene;1,5-dimethyl-bicyclo[3.2.1]octan-8-one oxime;4-(4-hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxaldehyde;3-buten-2-ol; 2-[[[2,4(or3,5)-dimethyl-3-cyclohexen-1-yl]methylene]amino]benzoic acid, methylester; 8-cyclohexadecen-1-one; methyl ionone; 2,6-dimethyl-7-octen-2-ol;2-methoxy-4-(2-propenyl)phenol; (2E)-3,7-dimethyl-2,6-Octadien-1-ol;2-hydroxy-Benzoic acid, (3Z)-3-hexenyl ester; 2-tridecenenitrile;4-(2,2-dimethyl-6-methylenecyclohexyl)-3-methyl-3-buten-2-one;tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran; Acetic acid,(2-methylbutoxy)-, 2-propenyl ester; Benzoic acid, 2-hydroxy-,3-methylbutyl ester; 2-Buten-1-one,1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-, (Z)-; Cyclopentanecarboxylicacid, 2-hexyl-3-oxo-, methyl ester; Benzenepropanal,4-ethyl-.alpha.,.alpha.-dimethyl-; 3-Cyclohexene-1-carboxaldehyde,3-(4-hydroxy-4-methylpentyl)-; Ethanone,1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)-,[3R-(3.alpha.,3a.beta.,7.beta.,8a.alpha.)]-; Undecanal,2-methyl-2H-Pyran-2-one, 6-butyltetrahydro-; Benzenepropanal,4-(1,1-dimethylethyl)-.alpha.-methyl-; 2(3H)-Furanone, 5-heptyldihydro-;Benzoic acid, 2-[(7-hydroxy-3,7-dimethyloctylidene)amino]-, methyl;Benzoic acid, 2-hydroxy-, phenylmethyl ester; Naphthalene, 2-methoxy-;2-Cyclopenten-1-one, 2-hexyl-; 2(3H)-Furanone, 5-hexyldihydro-;Oxiranecarboxylic acid, 3-methyl-3-phenyl-, ethyl ester;2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl-; Benzenepentanol,.gamma.-methyl-; 3-Octanol, 3,7-dimethyl-;3,7-dimethyl-2,6-octadienenitrile; 3,7-dimethyl-6-octen-1-ol; Terpineolacetate; 2-methyl-6-methylene-7-Octen-2-ol, dihydro derivative;3a,4,5,6,7,7a-hexahydro-4,7-Methano-1H-inden-6-ol propanoate;3-methyl-2-buten-1-ol acetate; (Z)-3-Hexen-1-ol acetate;2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol;4-(octahydro-4,7-methano-5H-inden-5-ylidene)-butanal;3-2,4-dimethyl-cyclohexene-1-carboxaldehyde;1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)-ethanone;2-hydroxy-benzoic acid, methyl ester; 2-hydroxy-benzoic acid, hexylester; 2-phenoxy-ethanol; 2-hydroxy-benzoic acid, pentyl ester;2,3-heptanedione; 2-hexen-1-ol; 6-Octen-2-ol, 2,6-dimethyl-; damascone(alpha, beta, gamma or delta or mixtures thereof),4,7-Methano-1H-inden-6-ol, 3a,4,5,6,7,7a-hexahydro-, acetate;9-Undecenal; 8-Undecenal; Isocyclocitral; Ethanone,1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)-;3-Cyclohexene-1-carboxaldehyde, 3,5-dimethyl-;3-Cyclohexene-1-carboxaldehyde, 2,4-dimethyl-; 1,6-Octadien-3-ol,3,7-dimethyl-; 1,6-Octadien-3-ol, 3,7-dimethyl-, acetate; Lilial(p-t-Bucinal), and Cyclopentanone,2-[2-(4-methyl-3-cyclohexen-1-yl)propyl]- and1-methyl-4-(1-methylethenyl)cyclohexene and mixtures thereof.

In one aspect the composition may comprise an encapsulated perfumeparticle comprising either a water-soluble hydroxylic compound ormelamine-formaldehyde or modified polyvinyl alcohol. In one aspect theencapsulate comprises (a) an at least partially water-soluble solidmatrix comprising one or more water-soluble hydroxylic compounds,preferably starch; and (b) a perfume oil encapsulated by the solidmatrix.

In a further aspect the perfume may be pre-complexed with a polyamine,preferably a polyethylenimine so as to form a Schiff base.

Polymers—The composition may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid co-polymers.

The composition may comprise one or more amphiphilic cleaning polymerssuch as the compound having the following general structure:bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)-bis((C₂H₅O)(C₂H₄O)n),wherein n=from 20 to 30, and x=from 3 to 8, or sulphated or sulphonatedvariants thereof.

The composition may comprise amphiphilic alkoxylated grease cleaningpolymers which have balanced hydrophilic and hydrophobic properties suchthat they remove grease particles from fabrics and surfaces. Specificembodiments of the amphiphilic alkoxylated grease cleaning polymers ofthe present invention comprise a core structure and a plurality ofalkoxylate groups attached to that core structure. These may comprisealkoxylated polyalkylenimines, preferably having an inner polyethyleneoxide block and an outer polypropylene oxide block.

Alkoxylated polycarboxylates such as those prepared from polyacrylatesare useful herein to provide additional grease removal performance. Suchmaterials are described in WO91/08281 and PCT90/01815. Chemically, thesematerials comprise polyacrylates having one ethoxy side-chain per every7-8 acrylate units. The side-chains are of the formula —(CH₂CH₂O)_(m)(CH₂)_(n)CH₃ wherein m is 2-3 and n is 6-12. The side-chains areester-linked to the polyacrylate “backbone” to provide a “comb” polymertype structure. The molecular weight can vary, but is typically in therange of 2000 to 50,000. Such alkoxylated polycarboxylates can comprisefrom 0.05 wt % to 10 wt % of the compositions herein.

The isoprenoid-derived surfactants of the present invention, and theirmixtures with other cosurfactants and other adjunct ingredients, areparticularly suited to be used with an amphilic graft co-polymer,preferably the amphilic graft co-polymer comprises (i) polyethyeleneglycol backbone; and (ii) and at least one pendant moiety selected frompolyvinyl acetate, polyvinyl alcohol and mixtures thereof. A preferredamphilic graft co-polymer is Sokalan HP22, supplied from BASF. Suitablepolymers include random graft copolymers, preferably a polyvinyl acetategrafted polyethylene oxide copolymer having a polyethylene oxidebackbone and multiple polyvinyl acetate side chains. The molecularweight of the polyethylene oxide backbone is preferably 6000 and theweight ratio of the polyethylene oxide to polyvinyl acetate is 40 to 60and no more than 1 grafting point per 50 ethylene oxide units.

Carboxylate polymer—The composition of the present invention may alsoinclude one or more carboxylate polymers such as a maleate/acrylaterandom copolymer or polyacrylate homopolymer. In one aspect, thecarboxylate polymer is a polyacrylate homopolymer having a molecularweight of from 4,000 to 9,000Da, or from 6,000 to 9,000Da.

Soil release polymer—The composition of the present invention may alsoinclude one or more soil release polymers having a structure as definedby one of the following structures (I), (II) or (III):

—[(OCHR¹—CHR²)_(a)—O—OC—Ar—CO—]_(d)   (I)

—[(OCHR³≧CHR⁴)_(b)≧O—OC-sAr—CO—]_(e)   (II)

—[(OCHR⁵—CHR⁶)_(c)—OR⁷]_(f)   (III)

wherein:

-   a, b and c are from 1 to 200;-   d, e and f are from 1 to 50;-   Ar is a 1,4-substituted phenylene;-   sAr is 1,3-substituted phenylene substituted in position 5 with    SO₃Me;-   Me is Li, K, Mg/2, Ca/2, Al/3, ammonium, mono-, di-, tri-, or    tetraalkylammonium wherein the alkyl groups are C₁-C₁₈ alkyl or    C₂-C₁₀ hydroxyalkyl, or mixtures thereof;-   R¹, R², R³, R⁴, R⁵ and R⁶ are independently selected from H or    C₁-C₁₈n- or iso-alkyl; and-   R⁷ is a linear or branched C₁-C₁₈ alkyl, or a linear or branched    C₂-C₃₀ alkenyl, or a cycloalkyl group with 5 to 9 carbon atoms, or a    C₈-C₃₀ aryl group, or a C₆-C₃₀ arylalkyl group.

Suitable soil release polymers are polyester soil release polymers suchas Repel-o-tex polymers, including Repel-o-tex, SF-2 and SRP6 suppliedby Rhodia. Other suitable soil release polymers include Texcarepolymers, including Texcare SRA100, SRA300, SRN100, SRN170, SRN240,SRN300 and SRN325 supplied by Clariant. Other suitable soil releasepolymers are Marloquest polymers, such as Marloquest SL supplied bySasol.

Cellulosic polymer—The composition of the present invention may alsoinclude one or more cellulosic polymers including those selected fromalkyl cellulose, alkyl alkoxyalkyl cellulose, carboxyalkyl cellulose,alkyl carboxyalkyl cellulose. In one aspect, the cellulosic polymers areselected from the group comprising carboxymethyl cellulose, methylcellulose, methyl hydroxyethyl cellulose, methyl carboxymethylcellulose, and mixures thereof. In one aspect, the carboxymethylcellulose has a degree of carboxymethyl substitution from 0.5 to 0.9 anda molecular weight from 100,000 to 300,000Da.

Enzymes—The composition may comprise one or more additional enzymeswhich provide cleaning performance and/or fabric care benefits. Examplesof suitable enzymes include, but are not limited to, hemicellulases,peroxidases, proteases, cellulases, xylanases, lipases, phospholipases,esterases, cutinases, pectinases, mannanases, pectate lyases,keratinases, reductases, oxidases, phenoloxidases, lipoxygenases,xanthanase, ligninases, pullulanases, tannases, pentosanases, malanases,β-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase,chlorophyllases and amylases, or mixtures thereof. A typical combinationis an enzyme cocktail that may comprise e.g. a protease and lipase inconjunction with amylase. When present in a composition, theaforementioned additional enzymes may be present at levels from 0.00001to 2 wt %, from 0.0001 to 1 wt % or from 0.001 to 0.5 wt % enzymeprotein by weight of the composition.

In general the properties of the selected enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Cellulases—Suitable cellulases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungalcellulases produced from Humicola insolens, Myceliophthora thermophilaand Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 andWO89/09259. Especially suitable cellulases are the alkaline or neutralcellulases having colour care benefits. Examples of such cellulases arecellulases described in EP0495257, EP0531372, WO96/11262, WO96/29397,and WO98/08940. Other examples are cellulase variants such as thosedescribed in WO94/07998, EP0531315, U.S. Pat. No. 5,457,046, U.S. Pat.No. 5,686,593, U.S. Pat. No. 5,763,254, WO95/24471, WO98/12307 andWO99/001544.

Other cellulases are endo-beta-1,4-glucanase enzyme having a sequence ofat least 97% identity to the amino acid sequence of position 1 toposition 773 of SEQ ID NO:2 of WO02/099091 or a family 44 xyloglucanase,which a xyloglucanase enzyme having a sequence of at least 60% identityto positions 40-559 of SEQ ID NO: 2 of WO01/062903.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes NS) Carezyme Premium™ (Novozymes NS), Celluclean™ (NovozymesNS), Celluclean Classic™ (Novozymes NS), Cellusoft™ (Novozymes NS),Whitezyme™ (Novozymes NS), Clazinase™, and Puradax HA™ (GenencorInternational Inc.), and KAC-500(B)™ (Kao Corporation).

Proteases—Suitable proteases include those of bacterial, fungal, plant,viral or animal origin e.g. vegetable or microbial origin. Microbialorigin is preferred. Chemically modified or protein engineered mutantsare included. It may be an alkaline protease, such as a serine proteaseor a metalloprotease. A serine protease may for example be of the 51family, such as trypsin, or the S8 family such as subtilisin. Ametalloproteases protease may for example be a thermolysin from e.g.family M4 or other metalloprotease such as those from M5, M7 or M8families.

The term “subtilases” refers to a sub-group of serine protease accordingto Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al.Protein Science 6 (1997) 501-523. Serine proteases are a subgroup ofproteases characterized by having a serine in the active site, whichforms a covalent adduct with the substrate. The subtilases may bedivided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitasefamily, the Proteinase K family, the Lantibiotic peptidase family, theKexin family and the Pyrolysin family.

Examples of subtilases are those derived from Bacillus such as Bacilluslentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacilluspumilus and Bacillus gibsonii described in; U.S. Pat. No. 7,262,042 andWO09/021867, and subtilisin lentus, subtilisin Novo, subtilisinCarlsberg, Bacillus licheniformis, subtilisin BPN′, subtilisin 309,subtilisin 147 and subtilisin 168 described in WO89/06279 and proteasePD138 described in (WO93/18140). Other useful proteases may be thosedescribed in WO92/175177, WO01/016285, WO02/026024 and WO02/016547.Examples of trypsin-like proteases are trypsin (e.g. of porcine orbovine origin) and the Fusarium protease described in WO89/06270,WO94/25583 and WO05/040372, and the chymotrypsin proteases derived fromCellumonas described in WO05/052161 and WO05/052146.

A further preferred protease is the alkaline protease from Bacilluslentus DSM 5483, as described for example in WO95/23221, and variantsthereof which are described in WO92/21760, WO95/23221, EP1921147 andEP1921148.

Examples of metalloproteases are the neutral metalloprotease asdescribed in WO07/044993 (Genencor Int.) such as those derived fromBacillus amyloliquefaciens.

Examples of useful proteases are the variants described in: WO92/19729,WO96/034946, WO98/20115, WO98/20116, WO99/011768, WO01/44452,WO03/006602, WO04/03186, WO04/041979, WO07/006305, WO11/036263,WO11/036264, especially the variants with substitutions in one or moreof the following positions: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130,160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235,236, 245, 248, 252 and 274 using the BPN′ numbering. More preferred thesubtilase variants may comprise the mutations: S3T, V41, S9R, A15T,K27R, *36D, V68A, N76D, N87S,R, *97E, A98S, S99G,D,A, S99AD, S101G,M,RS103A, V1041,Y,N, S106A, G118V,R, H120D,N, N123S, S128L, P129Q, S130A,G160D, Y167A, R170S, A194P, G195E, V199M, V2051, L217D, N218D, M222S,A232V, K235L, Q236H, Q245R, N252K, T274A (using BPN′ numbering).

Suitable commercially available protease enzymes include those soldunder the trade names Alcalase®, Duralase™, Durazym™, Relase®, Relase®Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®,Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra,Neutrase®, Everlase® and Esperase® (Novozymes NS), those sold under thetradename Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®,Preferenz™, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®,Properase®, Effectenz™, FN2®, FN3®, FN4®, Excellase®, Opticlean® andOptimase® (Danisco/DuPont), Axapem™ (Gist-Brocases N.V.), BLAP (sequenceshown in FIG. 29 of U.S. Pat. No. 5,352,604) and variants hereof (HenkelAG) and KAP (Bacillus alkalophilus subtilisin) from Kao.

Lipases and Cutinases—Suitable lipases and cutinases include those ofbacterial or fungal origin. Chemically modified or protein engineeredmutant enzymes are included. Examples include lipase from Thermomyces,e.g. from T. lanuginosus (previously named Humicola lanuginosa) asdescribed in EP258068 and EP305216, cutinase from Humicola, e.g. H.insolens (WO96/13580), lipase from strains of Pseudomonas (some of thesenow renamed to Burkholderia), e.g. P. alcaligenes or P.pseudoalcaligenes (EP218272), P. cepacia (EP331376), P. sp. strain SD705(WO95/06720 & WO96/27002), P. wisconsinensis (WO96/12012), GDSL-typeStreptomyces lipases (WO10/065455), cutinase from Magnaporthe grisea(WO10/107560), cutinase from Pseudomonas mendocina (U.S. Pat. No.5,389,536), lipase from Thermobifida fusca (WO11/084412), Geobacillusstearothermophilus lipase (WO11/084417), lipase from Bacillus subtilis(WO11/084599), and lipase from Streptomyces griseus (WO11/150157) and S.pristinaespiralis (WO12/137147).

Other examples are lipase variants such as those described in EP407225,WO92/05249, WO94/01541, WO94/25578, WO95/14783, WO95/30744, WO95/35381,WO95/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063,WO01/92502, WO07/87508 and WO09/109500.

Preferred commercial lipase products include include Lipolase™, Lipex™;Lipolex™ and Lipoclean™ (Novozymes NS), Lumafast (originally fromGenencor) and Lipomax (originally from Gist-Brocades).

Still other examples are lipases sometimes referred to asacyltransferases or perhydrolases, e.g. acyltransferases with homologyto Candida antarctica lipase A (WO10/111143), acyltransferase fromMycobacterium smegmatis (WO05/56782), perhydrolases from the CE 7 family(WO09/67279), and variants of the M. smegmatis perhydrolase inparticular the S54V variant used in the commercial product Gentle PowerBleach from Huntsman Textile Effects Pte Ltd (WO10/100028).

Amylases—Suitable amylases which can be used together with theenzyme/variant/blend of enzymes of the invention may be an alpha-amylaseor a glucoamylase and may be of bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included. Amylases include,for example, alpha-amylases obtained from Bacillus, e.g., a specialstrain of Bacillus licheniformis, described in more detail in GB1296839.

Suitable amylases include amylases having SEQ ID NO: 2 in WO95/10603 orvariants having 90% sequence identity to SEQ ID NO: 3 thereof. Preferredvariants are described in WO94/02597, WO94/18314, WO97/43424 and SEQ IDNO: 4 of WO99/019467, such as variants with substitutions in one or moreof the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156,178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264,304, 305, 391, 408, and 444.

Different suitable amylases include amylases having SEQ ID NO: 6 inWO02/010355 or variants thereof having 90% sequence identity to SEQ IDNO: 6. Preferred variants of SEQ ID NO: 6 are those having a deletion inpositions 181 and 182 and a substitution in position 193. Other amylaseswhich are suitable are hybrid alpha-amylase comprising residues 1-33 ofthe alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO:6 of WO06/066594 and residues 36-483 of the B. licheniformisalpha-amylase shown in SEQ ID NO: 4 of WO06/066594 or variants having90% sequence identity thereof. Preferred variants of this hybridalpha-amylase are those having a substitution, a deletion or aninsertion in one of more of the following positions: G48, T49, G107,H156, A181, N190, M197, 1201, A209 and Q264. Most preferred variants ofthe hybrid alpha-amylase comprising residues 1-33 of the alpha-amylasederived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO06/066594and residues 36-483 of SEQ ID NO: 4 are those having the substitutions:

-   M197T;-   H156Y+A181T+N190F+A209V+Q264S; or-   G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

Further amylases which are suitable are amylases having SEQ ID NO: 6 inWO99/019467 or variants thereof having 90% sequence identity to SEQ IDNO: 6. Preferred variants of SEQ ID NO: 6 are those having asubstitution, a deletion or an insertion in one or more of the followingpositions: R181, G182, H183, G184, N195, I206, E212, E216 and K269.Particularly preferred amylases are those having deletion in positionsR181 and G182, or positions H183 and G184.

Additional amylases which can be used are those having SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO96/023873 or variantsthereof having 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, adeletion or an insertion in one or more of the following positions: 140,181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476, using SEQID 2 of WO96/023873 for numbering. More preferred variants are thosehaving a deletion in two positions selected from 181, 182, 183 and 184,such as 181 and 182, 182 and 183, or positions 183 and 184. Mostpreferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7are those having a deletion in positions 183 and 184 and a substitutionin one or more of positions 140, 195, 206, 243, 260, 304 and 476.

Other amylases which can be used are amylases having SEQ ID NO: 2 ofWO08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90%sequence identity to SEQ ID NO: 2 of WO08/153815 or 90% sequenceidentity to SEQ ID NO: 10 in WO01/66712. Preferred variants of SEQ IDNO: 10 in WO01/66712 are those having a substitution, a deletion or aninsertion in one of more of the following positions: 176, 177, 178, 179,190, 201, 207, 211 and 264.

Further suitable amylases are amylases having SEQ ID NO: 2 ofWO09/061380 or variants having 90% sequence identity to SEQ ID NO: 2thereof. Preferred variants of SEQ ID NO: 2 are those having atruncation of the C-terminus and/or a substitution, a deletion or aninsertion in one of more of the following positions: Q87, Q98, S125,N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243,N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferredvariants of SEQ ID NO: 2 are those having the substitution in one ofmore of the following positions: Q87E,R, Q98R, S125A, N128C, T131I,T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R,R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180and/or S181 or of T182 and/or G183. Most preferred amylase variants ofSEQ ID NO: 2 are those having the substitutions:

-   N128C+K178L+T182G+Y305R+G475K;-   N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;-   S125A+N128C+K178L+T182G+Y305R+G475K; or-   S125A+N128C+T1311+T1651+K178L+T182G+Y305R+G475K wherein the variants    are C-terminally truncated and optionally further comprises a    substitution at position 243 and/or a deletion at position 180    and/or position 181.

Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 inWO01/66712 or a variant having at least 90% sequence identity to SEQ IDNO: 12. Preferred amylase variants are those having a substitution, adeletion or an insertion in one of more of the following positions ofSEQ ID NO: 12 in WO01/66712: R28, R118, N174; R181, G182, D183, G184,G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320,H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484.Particular preferred amylases include variants having a deletion of D183and G184 and having the substitutions R118K, N195F, R320K and R458K, anda variant additionally having substitutions in one or more positionselected from the group: M9, G149, G182, G186, M202, T257, Y295, N299,M323, E345 and A339, most preferred a variant that additionally hassubstitutions in all these positions.

Other examples are amylase variants such as those described inWO2011/098531, WO2013/001078 and WO2013/001087.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™,Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™ (fromNovozymes NS), and Rapidase™, Purastar™/Effectenz™, Powerase andPreferenz S100 (from Genencor International Inc./DuPont).

Peroxidases/Oxidases—Suitable peroxidases according to the invention isa peroxidase enzyme comprised by the enzyme classification EC 1.11.1.7,as set out by the Nomenclature Committee of the International Union ofBiochemistry and Molecular Biology (IUBMB), or any fragment derivedtherefrom, exhibiting peroxidase activity.

Suitable peroxidases include those of plant, bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful peroxidases include peroxidases from Coprinopsis, e.g., fromC. cinerea (EP179486), and variants thereof as those described inWO93/24618, WO95/10602, and WO98/15257.

A peroxidase also includes a haloperoxidase enzyme, such aschloroperoxidase, bromoperoxidase and compounds exhibitingchloroperoxidase or bromoperoxidase activity. Haloperoxidases areclassified according to their specificity for halide ions.Chloroperoxidases (E.C. 1.11.1.10) catalyze formation of hypochloritefrom chloride ions.

In an embodiment, the haloperoxidase of the invention is achloroperoxidase. Preferably, the haloperoxidase is a vanadiumhaloperoxidase, i.e., a vanadate-containing haloperoxidase. In apreferred method of the present invention the vanadate-containinghaloperoxidase is combined with a source of chloride ion.

Haloperoxidases have been isolated from many different fungi, inparticular from the fungus group dematiaceous hyphomycetes, such asCaldariomyces, e.g., C. fumago, Alternaria, Curvularia, e.g., C.verruculosa and C. inaequalis, Drechslera, Ulocladium and Botrytis.

Haloperoxidases have also been isolated from bacteria such asPseudomonas, e.g., P. pyrrocinia and Streptomyces, e.g., S.aureofaciens.

In an preferred embodiment, the haloperoxidase is derivable fromCurvularia sp., in particular Curvularia verruculosa or Curvulariainaequalis, such as C. inaequalis CBS 102.42 as described in WO95/27046;or C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 as describedin WO97/04102; or from Drechslera hartlebii as described in WO01/79459,Dendryphiella salina as described in WO01/79458, Phaeotrichoconiscrotalarie as described in WO01/79461, or Geniculosporium sp. asdescribed in WO01/79460.

An oxidase according to the invention include, in particular, anylaccase enzyme comprised by the enzyme classification EC 1.10.3.2, orany fragment derived therefrom exhibiting laccase activity, or acompound exhibiting a similar activity, such as a catechol oxidase (EC1.10.3.1), an o-aminophenol oxidase (EC 1.10.3.4), or a bilirubinoxidase (EC 1.3.3.5).

Preferred laccase enzymes are enzymes of microbial origin. The enzymesmay be derived from plants, bacteria or fungi (including filamentousfungi and yeasts).

Suitable examples from fungi include a laccase derivable from a strainof Aspergillus, Neurospora, e.g., N. crassa, Podospora, Botrytis,Collybia, Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T.versicolor, Rhizoctonia, e.g., R. solani, Coprinopsis, e.g., C. cinerea,C. comatus, C. friesii, and C. plicatilis, Psathyrella, e.g., P.condelleana, Panaeolus, e.g., P. papilionaceus, Myceliophthora, e.g., M.thermophila, Schytalidium, e.g., S. thermophilum, Polyporus, e.g., P.pinsitus, Phlebia, e.g., P. radiata (WO92/01046), or Coriolus, e.g., C.hirsutus (JP2238885).

Suitable examples from bacteria include a laccase derivable from astrain of Bacillus.

A laccase derived from Coprinopsis or Myceliophthora is preferred; inparticular a laccase derived from Coprinopsis cinerea, as disclosed inWO97/08325; or from Myceliophthora thermophila, as disclosed inWO95/33836.

Other preferred enzymes include pectate lyases sold under the tradenamesPectawash®, Pectaway®, Xpect® and mannanases sold under the tradenamesMannaway® (Novozymes), and Purabrite® (Danisco/Dupont).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated, for example, as a granulate, liquid, slurry, etc.Preferred detergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g. as disclosed in U.S. Pat.No. 4,106,991 and U.S. Pat. No. 4,661,452 and may optionally be coatedby methods known in the art. Examples of waxy coating materials arepoly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molarweights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50ethylene oxide units; ethoxylated fatty alcohols in which the alcoholcontains from 12 to 20 carbon atoms and in which there are 15 to 80ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591.Liquid enzyme preparations may, for instance, be stabilized by adding apolyol such as propylene glycol, a sugar or sugar alcohol, lactic acidor boric acid according to established methods. Protected enzymes may beprepared according to the method disclosed in EP238216.

Dye Transfer Inhibiting Agents—The compositions of the present inventionmay also include one or more dye transfer inhibiting agents. Suitablepolymeric dye transfer inhibiting agents include, but are not limitedto, polyvinylpyrrolidone polymers, polyamine N-oxide polymers,copolymers of N-vinylpyrrolidone and N-vinylimidazole,polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. Whenpresent in a composition, the dye transfer inhibiting agents may bepresent at levels from 0.0001 to 10 wt %, from 0.01 to 5 wt % or from0.1 to 3 wt %.

Brighteners—The compositions of the present invention can also containadditional components that may tint articles being cleaned, such asfluorescent brighteners. The composition may comprise C.I. fluorescentbrightener 260 in alpha-crystalline form having the following structure:

In one aspect, the brightener is a cold water soluble brightener, suchas the C.I. fluorescent brightener 260 in alpha-crystalline form. In oneaspect the brightener is predominantly in alpha-crystalline form, whichmeans that typically at least 50 wt %, at least 75 wt %, at least 90 wt%, at least 99 wt %, or even substantially all, of the C.I. fluorescentbrightener 260 is in alpha-crystalline form.

The brightener is typically in micronized particulate form, having aweight average primary particle size of from 3 to 30 micrometers, from 3micrometers to 20 micrometers, or from 3 to 10 micrometers.

The composition may comprise C.I. fluorescent brightener 260 inbeta-crystalline form, and the weight ratio of: (i) C.I. fluorescentbrightener 260 in alpha-crystalline form, to (ii) C.I. fluorescentbrightener 260 in beta-crystalline form may be at least 0.1, or at least0.6. BE680847 relates to a process for making 0.1 fluorescent brightener260 in alpha-crystalline form.

Commercial optical brighteners which may be useful in the presentinvention can be classified into subgroups, which include, but are notnecessarily limited to, derivatives of stilbene, pyrazoline, coumarin,carboxylic acid, methinecyanines, dibenzothiophene-5,5-dioxide, azoles,5- and 6-membered-ring heterocycles, and other miscellaneous agents.Examples of such brighteners are disclosed in “The Production andApplication of Fluorescent Brightening Agents”, M. Zahradnik, Publishedby John Wiley & Sons, New York (1982). Specific nonlimiting examples ofoptical brighteners which are useful in the present compositions arethose identified in U.S. Pat. No. 4,790,856 and U.S. Pat. No. 3,646,015.

A further suitable brightener has the structure below:

Suitable fluorescent brightener levels include lower levels of from 0.01wt %, from 0.05 wt %, from 0.1 wt % or from 0.2 wt % to upper levels of0.5 wt % or 0.75 wt %.

In one aspect the brightener may be loaded onto a clay to form aparticle.Silicate salts - The compositions of the present invention canalso contain silicate salts, such as sodium or potassium silicate. Thecomposition may comprise of from 0 wt % to less than 10 wt % silicatesalt, to 9 wt %, or to 8 wt %, or to 7 wt %, or to 6 wt %, or to 5 wt %,or to 4 wt %, or to 3 wt %, or even to 2 wt %, and from above 0 wt %, orfrom 0.5 wt %, or from 1 wt % silicate salt. A suitable silicate salt issodium silicate.

Dispersants—The compositions of the present invention can also containdispersants. Suitable water-soluble organic materials include the homo-or co-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms.

Enzyme Stabilizers—Enzymes for use in compositions can be stabilized byvarious techniques. The enzymes employed herein can be stabilized by thepresence of water-soluble sources of calcium and/or magnesium ions.Examples of conventional stabilizing agents are, e.g. a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g. an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in, for example,WO92/19709 and WO92/19708 In case of aqueous compositions comprisingprotease, a reversible protease inhibitor, such as a boron compoundincluding borate, 4-formyl phenylboronic acid, phenylboronic acid andderivatives thereof, or compounds such as calcium formate, sodiumformate and 1,2-propane diol can be added to further improve stability.

Solvents—Suitable solvents include water and other solvents such aslipophilic fluids. Examples of suitable lipophilic fluids includesiloxanes, other silicones, hydrocarbons, glycol ethers, glycerinederivatives such as glycerine ethers, perfluorinated amines,perfluorinated and hydrofluoroether solvents, low-volatilitynonfluorinated organic solvents, diol solvents, otherenvironmentally-friendly solvents and mixtures thereof.

Structurant/Thickeners—Structured liquids can either be internallystructured, whereby the structure is formed by primary ingredients (e.g.surfactant material) and/or externally structured by providing a threedimensional matrix structure using secondary ingredients (e.g. polymers,clay and/or silicate material). The composition may comprise astructurant, from 0.01 to 5 wt %, or from 0.1 to 2.0 wt %. Thestructurant is typically selected from the group consisting ofdiglycerides and triglycerides, ethylene glycol distearate,microcrystalline cellulose, cellulose-based materials, microfibercellulose, hydrophobically modified alkali-swellable emulsions such asPolygel W30 (3VSigma), biopolymers, xanthan gum, gellan gum, andmixtures thereof. A suitable structurant includes hydrogenated castoroil, and non-ethoxylated derivatives thereof. A suitable structurant isdisclosed in US6855680. Such structurants have a thread-like structuringsystem having a range of aspect ratios. Other suitable structurants andthe processes for making them are described in WO10/034736.

Conditioning Agents—The composition of the present invention may includea high melting point fatty compound. The high melting point fattycompound useful herein has a melting point of 25° C. or higher, and isselected from the group consisting of fatty alcohols, fatty acids, fattyalcohol derivatives, fatty acid derivatives, and mixtures thereof. Suchcompounds of low melting point are not intended to be included in thissection. Non-limiting examples of the high melting point compounds arefound in International Cosmetic Ingredient Dictionary, Fifth Edition,1993, and CTFA Cosmetic Ingredient Handbook, Second Edition, 1992.

The high melting point fatty compound is included in the composition ata level of from 0.1 to 40 wt %, from 1 to 30 wt %, from 1.5 to 16 wt %,from 1.5 to 8 wt % in view of providing improved conditioning benefitssuch as slippery feel during the application to wet hair, softness andmoisturized feel on dry hair.

The compositions of the present invention may contain a cationicpolymer. Concentrations of the cationic polymer in the compositiontypically range from 0.05 to 3 wt %, from 0.075 to 2.0 wt %, or from 0.1to 1.0 wt %. Suitable cationic polymers will have cationic chargedensities of at least 0.5 meq/gm, at least 0.9 meq/gm, at least 1.2meq/gm, at least 1.5 meq/gm, or less than 7 meq/gm, and less than 5meq/gm, at the pH of intended use of the composition, which pH willgenerally range from pH3 to pH9, or between pH4 and pH8. Herein,“cationic charge density” of a polymer refers to the ratio of the numberof positive charges on the polymer to the molecular weight of thepolymer. The average molecular weight of such suitable cationic polymerswill generally be between 10,000 and 10 million, between 50,000 and 5million, or between 100,000 and 3 million.

Suitable cationic polymers for use in the compositions of the presentinvention contain cationic nitrogen-containing moieties such asquaternary ammonium or cationic protonated amino moieties. Any anioniccounterions can be used in association with the cationic polymers solong as the polymers remain soluble in water, in the composition, or ina coacervate phase of the composition, and so long as the counterionsare physically and chemically compatible with the essential componentsof the composition or do not otherwise unduly impair compositionperformance, stability or aesthetics. Nonlimiting examples of suchcounterions include halides (e.g., chloride, fluoride, bromide, iodide),sulfate and methylsulfate.

Nonlimiting examples of such polymers are described in the CTFA CosmeticIngredient Dictionary, 3rd edition, edited by Estrin, Crosley, andHaynes, (The Cosmetic, Toiletry, and Fragrance Association, Inc.,Washington, D.C. (1982)).

Other suitable cationic polymers for use in the composition includepolysaccharide polymers, cationic guar gum derivatives, quaternarynitrogen-containing cellulose ethers, synthetic polymers, copolymers ofetherified cellulose, guar and starch. When used, the cationic polymersherein are either soluble in the composition or are soluble in a complexcoacervate phase in the composition formed by the cationic polymer andthe anionic, amphoteric and/or zwitterionic surfactant componentdescribed hereinbefore. Complex coacervates of the cationic polymer canalso be formed with other charged materials in the composition. Suitablecationic polymers are described in U.S. Pat. No. 3,962,418; U.S. Pat.No. 3,958,581; and US2007/0207109.

The composition of the present invention may include a nonionic polymeras a conditioning agent. Polyalkylene glycols having a molecular weightof more than 1000 are useful herein. Useful are those having thefollowing general formula:

wherein R⁹⁵ is selected from the group consisting of H, methyl, andmixtures thereof. Conditioning agents, and in particular silicones, maybe included in the composition. The conditioning agents useful in thecompositions of the present invention typically comprise a waterinsoluble, water dispersible, non-volatile, liquid that formsemulsified, liquid particles. Suitable conditioning agents for use inthe composition are those conditioning agents characterized generally assilicones (e.g., silicone oils, cationic silicones, silicone gums, highrefractive silicones, and silicone resins), organic conditioning oils(e.g., hydrocarbon oils, polyolefins, and fatty esters) or combinationsthereof, or those conditioning agents which otherwise form liquid,dispersed particles in the aqueous surfactant matrix herein. Suchconditioning agents should be physically and chemically compatible withthe essential components of the composition, and should not otherwiseunduly impair composition stability, aesthetics or performance.

The concentration of the conditioning agent in the composition should besufficient to provide the desired conditioning benefits. Suchconcentration can vary with the conditioning agent, the conditioningperformance desired, the average size of the conditioning agentparticles, the type and concentration of other components, and otherlike factors.

The concentration of the silicone conditioning agent typically rangesfrom 0.01 to 10 wt %. Non-limiting examples of suitable siliconeconditioning agents, and optional suspending agents for the silicone,are described in U.S. Reissue Pat. No. 34,584; U.S. Pat. No. 5,104,646;U.S. Pat. No. 5,106,609; U.S. Pat. No. 4,152,416; U.S. Pat. No.2,826,551; U.S. Pat. No. 3,964,500; U.S. Pat. No. 4,364,837; U.S. Pat.No. 6,607,717; U.S. Pat. No. 6,482,969; U.S. Pat. No. 5,807,956; U.S.Pat. No. 5,981,681; U.S. Pat. No. 6,207,782; U.S. Pat. No. 7,465,439;U.S. Pat. No. 7,041,767; U.S. Pat. No. 7,217,777; U.S. Pat. No.2007/0286837A1; US2005/0048549A1; US2007/0041929A1; GB849433;DE10036533, which are all incorporated herein by reference; Chemistryand Technology of Silicones, New York: Academic Press (1968); GeneralElectric Silicone Rubber Product Data Sheets SE 30, SE 33, SE 54 and SE76; Silicon Compounds, Petrarch Systems, Inc. (1984); and inEncyclopedia of Polymer Science and Engineering, vol. 15, 2d ed., pp204-308, John Wiley & Sons, Inc. (1989).

The compositions of the present invention may also comprise from 0.05 to3 wt % of a at least one organic conditioning oil as the conditioningagent, either alone or in combination with other conditioning agents,such as the silicones (described herein). Suitable conditioning oilsinclude hydrocarbon oils, polyolefins, and fatty esters. Also suitablefor use in the compositions herein are the conditioning agents describedin U.S. Pat. No. 5,674,478 and U.S. Pat. No. 5,750,122 or in U.S. Pat.No. 4,529,586; U.S. Pat. No. 4,507,280; U.S. Pat. No. 4,663,158; U.S.Pat. No. 4,197,865; U.S. Pat. No. 4,217,914; U.S. Pat. No. 4,381,919;and U.S. Pat. No. 4,422,853.

Hygiene and malodour—The compositions of the present invention may alsocomprise one or more of zinc ricinoleate, thymol, quaternary ammoniumsalts such as Bardac®, polyethylenimines (such as Lupasol® from BASF)and zinc complexes thereof, silver and silver compounds, especiallythose designed to slowly release Ag⁺ or nano-silver dispersions.

Probiotics—The compositions may comprise probiotics such as thosedescribed in WO09/043709.

Suds Boosters—If high sudsing is desired, suds boosters such as theC₁₀-C₁₆ alkanolamides or C₁₀-C₁₄ alkyl sulphates can be incorporatedinto the compositions, typically at 1 to 10 wt % levels. The C₁₀-C₁₄monoethanol and diethanol amides illustrate a typical class of such sudsboosters. Use of such suds boosters with high sudsing adjunctsurfactants such as the amine oxides, betaines and sultaines noted aboveis also advantageous. If desired, water-soluble magnesium and/or calciumsalts such as MgCl₂, MgSO₄, CaCl₂, CaSO₄ and the like, can be added atlevels of, typically, 0.1 to 2 wt %, to provide additional suds and toenhance grease removal performance.

Suds Suppressors—Compounds for reducing or suppressing the formation ofsuds can be incorporated into the compositions of the present invention.Suds suppression can be of particular importance in the so-called “highconcentration cleaning process” as described in U.S. Pat. No. 4,489,455and U.S. Pat. No. 4,489,574, and in front-loading-style washingmachines. A wide variety of materials may be used as suds suppressors,and suds suppressors are well known to those skilled in the art. Seee.g. Kirk Othmer Encyclopedia of Chemical Technology, Third Edition,Volume 7, p.430-447 (John Wiley & Sons, Inc., 1979). Examples of sudssupressors include monocarboxylic fatty acid and soluble salts therein,high molecular weight hydrocarbons such as paraffin, fatty acid esters(e.g., fatty acid triglycerides), fatty acid esters of monovalentalcohols, aliphatic C₁₈-C₄₀ ketones (e.g., stearone), N-alkylated aminotriazines, waxy hydrocarbons preferably having a melting point belowabout 100° C., silicone suds suppressors, and secondary alcohols. Sudssupressors are described in U.S. Pat. No. 2,954,347; U.S. Pat. No.4,265,779; U.S. Pat. No. 4,265,779; U.S. Pat. No. 3,455,839; U.S. Pat.No. 3,933,672; U.S. Pat. No. 4,652,392; U.S. Pat. No. 4,978,471; U.S.Pat. No. 4,983,316; U.S. Pat. No. 5,288,431; U.S. Pat. No. 4,639,489;U.S. Pat. No. 4,749,740; U.S. Pat. No. 4,798,679; U.S. Pat. No.4,075,118; EP89307851.9; EP150872; and DOS 2,124,526.

For any detergent compositions to be used in automatic laundry washingmachines, suds should not form to the extent that they overflow thewashing machine. Suds suppressors, when utilized, are preferably presentin a “suds suppressing amount. By “suds suppressing amount” is meantthat the formulator of the composition can select an amount of this sudscontrolling agent that will sufficiently control the suds to result in alow-sudsing laundry detergent for use in automatic laundry washingmachines.

The compositions herein will generally comprise from 0 to 10 wt % ofsuds suppressor. When utilized as suds suppressors, monocarboxylic fattyacids, and salts therein, will be present typically in amounts up to 5wt %. Preferably, from 0.5 to 3 wt % of fatty monocarboxylate sudssuppressor is utilized. Silicone suds suppressors are typically utilizedin amounts up to 2.0 wt %, although higher amounts may be used.Monostearyl phosphate suds suppressors are generally utilized in amountsranging from 0.1 to 2 wt %. Hydrocarbon suds suppressors are typicallyutilized in amounts ranging from 0.01 to 5.0 wt %, although higherlevels can be used. The alcohol suds suppressors are typically used at0.2 to 3 wt %.

The compositions herein may have a cleaning activity over a broad rangeof pH. In certain embodiments the compositions have cleaning activityfrom pH4 to pH11.5. In other embodiments, the compositions are activefrom pH6 to pH11, from pH7 to pH11, from pH8 to pH11, from pH9 to pH11,or from pH10 to pH11.5.

The compositions herein may have cleaning activity over a wide range oftemperatures, e.g., from 10° C. or lower to 90° C. Preferably thetemperature will be below 50° C. or 40° C. or even 30° C. In certainembodiments, the optimum temperature range for the compositions is from10° C. to 20° C., from 15° C. to 25° C., from 15° C. to 30° C., from 20°C. to 30° C., from 25° C. to 35° C., from 30° C. to 40° C., from 35° C.to 45° C., or from 40° C. to 50° C.

Form of the Composition

The compositions described herein are advantageously employed forexample, in laundry applications, hard surface cleaning, dishwashingapplications, as well as cosmetic applications such as dentures, teeth,hair and skin. The compositions of the invention are in particular solidor liquid cleaning and/or treatment compositions. In one aspect theinvention relates to a composition, wherein the form of the compositionis selected from the group consisting of a regular, compact orconcentrated liquid; a gel; a paste; a soap bar; a regular or acompacted powder; a granulated solid; a homogenous or a multilayertablet with two or more layers (same or different phases); a pouchhaving one or more compartments; a single or a multi-compartment unitdose form; or any combination thereof.

The form of the composition may separate the components physically fromeach other in compartments such as e.g. water dissolvable pouches or indifferent layers of tablets. Thereby negative storage interactionbetween components can be avoided. Different dissolution profiles ofeach of the compartments can also give rise to delayed dissolution ofselected components in the wash solution.

Pouches can be configured as single or multicompartments. It can be ofany form, shape and material which is suitable for hold the composition,e.g. without allowing the release of the composition to release of thecomposition from the pouch prior to water contact. The pouch is madefrom water soluble film which encloses an inner volume. Said innervolume can be divided into compartments of the pouch. Preferred filmsare polymeric materials preferably polymers which are formed into a filmor sheet. Preferred polymers, copolymers or derivates thereof areselected polyacrylates, and water soluble acrylate copolymers, methylcellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose,hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin,poly methacrylates, most preferably polyvinyl alcohol copolymers and,hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymerin the film for example PVA is at least about 60%. Preferred averagemolecular weight will typically be about 20,000 to about 150,000. Filmscan also be of blended compositions comprising hydrolytically degradableand water soluble polymer blends such as polylactide and polyvinylalcohol (known under the Trade reference M8630 as sold by MonoSol LLC,Indiana, USA) plus plasticisers like glycerol, ethylene glycerol,propylene glycol, sorbitol and mixtures thereof. The pouches cancomprise a solid laundry cleaning composition or part components and/ora liquid cleaning composition or part components separated by the watersoluble film. The compartment for liquid components can be different incomposition than compartments containing solids (US2009/0011970 A1).

Lipase Particles

The lipase variants comprised in the water-soluble film of the inventionmay be present as lipase particles. The lipase particles may evencontain one or more Additional Enzymes, as described below.

Lipase particles are any form of lipase variant in a solid particulateform. That can be as lipase crystals, lipase precipitate, spray orfreeze-dried lipase or any form of granulated lipase, either as a powderor a suspension in liquid. Typically the particle size, measured asequivalent spherical diameter (volume based average particle size), ofthe lipase particles is below 2 mm, preferably below 1 mm, below 0.5 mm,below 0.25 mm, or below 0.1 mm; and above 0.05 μm, preferably above 0.1μm, above 0.5 μm, above 1 μm, above 5 μm or above 10 μm.

In a preferred embodiment, the particle size of the lipase particles isfrom 0.5 μm to 100 μm.

The lipase particles contain at least 1% w/w lipase protein, preferablyat least 5% w/w lipase protein, at least 10% w/w lipase protein, atleast 20% w/w lipase protein, at least 30% w/w lipase protein, at least40% w/w lipase protein, at least 50% w/w lipase protein, at least 60%w/w lipase protein, at least 70% w/w lipase protein, at least 80% w/wlipase protein, or at least 90% w/w lipase protein.

In a preferred embodiment, the lipase particles are lipase crystals, orthe lipase protein is on a crystalline form.

Enzyme crystallization may be carried out in a number of ways, as knownin the art (e.g., as described in WO91/09943 or WO94/22903).

The lipase may be formulated in the lipase particle as known in the artfor solid enzyme formulations, such as formulations for reducing dust,improving stability and/or modifying relase rate of the enzyme. Thelipase particle may also be formulated in a matrix or coated with agentssuppressing dissolution of the enzyme particle in the PVOH/film solutionused for preparing the water-soluble film.

The lipase molecules on the surface of the lipase particles may also becross-linked, like CLECs (Cross-Linked Enzyme Crystals) or CLEA(Cross-Linked Enzyme Aggregate).

Water-Soluble Film

Water-soluble films, optional ingredients for use therein, and methodsof making the same are well known in the art. In one class ofembodiments, the water-soluble film includes PVOH. PVOH is a syntheticresin generally prepared by the alcoholysis, usually termed hydrolysisor saponification, of polyvinyl acetate. Fully hydrolyzed PVOH, whereinvirtually all the acetate groups have been converted to alcohol groups,is a strongly hydrogen-bonded, highly crystalline polymer whichdissolves only in hot water—greater than about 140° F. (60° C.). If asufficient number of acetate groups are allowed to remain after thehydrolysis of polyvinyl acetate, the PVOH polymer then being known aspartially hydrolyzed, it is more weakly hydrogen-bonded and lesscrystalline and is soluble in cold water—less than about 50° F. (10°C.). An intermediate cold/hot water-soluble film can include, forexample, intermediate partially-hydrolyzed PVOH (e.g., with degrees ofhydrolysis of about 94% to about 98%), and is readily soluble only inwarm water—e.g., rapid dissolution at temperatures of about 40° C. andgreater. Both fully and partially hydrolyzed PVOH types are commonlyreferred to as PVOH homopolymers although the partially hydrolyzed typeis technically a vinyl alcohol-vinyl acetate copolymer.

The degree of hydrolysis of the PVOH included in the water-soluble filmsof the present disclosure can be about 75% to about 99%. As the degreeof hydrolysis is reduced, a film made from the resin will have reducedmechanical strength but faster solubility at temperatures below about20° C. As the degree of hydrolysis increases, a film made from the resinwill tend to be mechanically stronger and the thermoformability willtend to decrease. The degree of hydrolysis of the PVOH can be chosensuch that the water-solubility of the resin is temperature dependent,and thus the solubility of a film made from the resin, compatibilizingagent, and additional ingredients is also influenced. In one class ofembodiments the film is cold water-soluble. A cold water-soluble film,soluble in water at a temperature of less than 10° C., can include PVOHwith a degree of hydrolysis in a range of about 75% to about 90%, or ina range of about 80% to about 90%, or in a range of about 85% to about90%. In another class of embodiments the film is hot water-soluble. Ahot water-soluble film, soluble in water at a temperature of at leastabout 60° C., can include PVOH with a degree of hydrolysis of at leastabout 98%.

Other film-forming resins for use in addition to or in an alternative toPVOH can include, but are not limited to, modified polyvinyl alcohols,polyacrylates, water-soluble acrylate copolymers, polyacrylates,polyacryamides, polyvinyl pyrrolidone, pullulan, water-soluble naturalpolymers including, but not limited to, guar gum, xanthan gum,carrageenan, and starch, water-soluble polymer derivatives including,but not limited to, ethoxylated starch and hydroxypropylated starch,poly(sodium acrylamido-2-methylpropane sulfonate),polymonomethylmaleate, copolymers thereof, and combinations of any ofthe foregoing. In one class of embodiments, the film-forming resin is aterpolymer consisting of vinyl alcohol, vinyl acetate, and sodiumacrylamido-2-methylpropanesulfonate. Unexpectedly, water-soluble filmsbased on a vinyl alcohol, vinyl acetate, and sodiumacrylamido-2-methylpropanesulfonate terpolymer have demonstrated a highpercent recovery of enzyme.

The water-soluble resin can be included in the water-soluble film in anysuitable amount, for example an amount in a range of about 35 wt % toabout 90 wt %. The preferred weight ratio of the amount of thewater-soluble resin as compared to the combined amount of all enzymes,enzyme stabilizers, and secondary additives can be any suitable ratio,for example a ratio in a range of about 0.5 to about 5, or about 1 to 3,or about 1 to 2.

Water-soluble resins for use in the films described herein (including,but not limited to PVOH resins) can be characterized by any suitableviscosity for the desired film properties, optionally a viscosity in arange of about 5.0 to about 30.0 cP, or about 10.0 cP to about 25 cP.The viscosity of a PVOH resin is determined by measuring a freshly madesolution using a Brookfield LV type viscometer with UL adapter asdescribed in British Standard EN ISO 15023-2:2006 Annex E BrookfieldTest method. It is international practice to state the viscosity of 4%aqueous polyvinyl alcohol solutions at 20° C. All PVOH viscositiesspecified herein in cP should be understood to refer to the viscosity of4% aqueous polyvinyl alcohol solution at 20° C., unless specifiedotherwise.

It is well known in the art that the viscosity of a PVOH resin iscorrelated with the weight average molecular weight (Mw) of the samePVOH resin, and often the viscosity is used as a proxy for Mw. Thus, theweight average molecular weight of the water-soluble resin optionallycan be in a range of about 35,000 to about 190,000, or about 80,000 toabout 160,000. The molecular weight of the resin need only be sufficientto enable it to be molded by suitable techniques to form a thin plasticfilm.

The water-soluble films according to the present disclosure may includeother optional additive ingredients including, but not limited to,plasticizers, surfactants, defoamers, film formers, antiblocking agents,internal release agents, anti-yellowing agents and other functionalingredients, for example in amounts suitable for their intended purpose.

Water is recognized as a very efficient plasticizer for PVOH and otherpolymers; however, the volatility of water makes its utility limitedsince polymer films need to have at least some resistance (robustness)to a variety of ambient conditions including low and high relativehumidity. Glycerin is much less volatile than water and has been wellestablished as an effective plasticizer for PVOH and other polymers.Glycerin or other such liquid plasticizers by themselves can causesurface “sweating” and greasiness if the level used in the filmformulation is too high. This can lead to problems in a film such asunacceptable feel to the hand of the consumer and even blocking of thefilm on the roll or in stacks of sheets if the sweating is not mitigatedin some manner, such as powdering of the surface. This could becharacterized as over plasticization. However, if too little plasticizeris added to the film the film may lack sufficient ductility andflexibility for many end uses, for example to be converted into a finaluse format such as pouches.

Plasticizers for use in water-soluble films of the present disclosureinclude, but are not limited to, sorbitol, glycerol, diglycerol,propylene glycol, ethylene glycol, diethyleneglycol, triethylene glycol,tetraethyleneglycol, polyethylene glycols up to MW 400, 2 methyl 1,3propane diol, lactic acid, monoacetin, triacetin, triethyl citrate,1,3-butanediol, trimethylolpropane (TMP), polyether triol, andcombinations thereof. Polyols, as described above, are generally usefulas plasticizers. As less plasticizer is used, the film can become morebrittle, whereas as more plasticizer is used the film can lose tensilestrength. Plasticizers can be included in the water-soluble films in anamount in a range of about 25 phr to about 50 phr, or from about 30 phrto about 45 phr, or from about 32 phr to about 42 phr, for example.

Surfactants for use in water-soluble films are well known in the art.Optionally, surfactants are included to aid in the dispersion of theresin solution upon casting. Suitable surfactants for water-solublefilms of the present disclosure include, but are not limited to, dialkylsulfosuccinates, lactylated fatty acid esters of glycerol and propyleneglycol, lactylic esters of fatty acids, sodium alkyl sulfates,polysorbate 20, polysorbate 60, polysorbate 65, polysorbate 80, alkylpolyethylene glycol ethers, lecithin, acetylated fatty acid esters ofglycerol and propylene glycol, sodium lauryl sulfate, acetylated estersof fatty acids, myristyl dimethylamine oxide, trimethyl tallow alkylammonium chloride, quaternary ammonium compounds, salts thereof andcombinations of any of the forgoing. Thus, surfactants can be includedin the water-soluble films in an amount of less than about 2 phr, forexample less than about 1 phr, or less than about 0.5 phr, for example.

One type of secondary component contemplated for use is a defoamer.Defoamers can aid in coalescing of foam bubbles. Suitable defoamers foruse in water-soluble films according to the present disclosure include,but are not limited to, hydrophobic silicas, for example silicon dioxideor fumed silica in fine particle sizes, including Foam Blast® defoamersavailable from Emerald Performance Materials, including Foam Blast® 327,Foam Blast® UVD, Foam Blast® 163, Foam Blast® 269, Foam Blast® 338, FoamBlast® 290, Foam Blast® 332, Foam Blast® 349, Foam Blast® 550 and FoamBlast® 339, which are proprietary, non-mineral oil defoamers. Inembodiments, defoamers can be used in an amount of 0.5 phr, or less, forexample, 0.05 phr, 0.04 phr, 0.03 phr, 0.02 phr, or 0.01 phr.Preferably, significant amounts of silicon dioxide will be avoided, inorder to avoid stress whitening.

Processes for making water-soluble articles, including films, includecasting, blow-molding, extrusion and blown extrusion, as known in theart. One contemplated class of embodiments is characterized by thewater-soluble film described herein being formed by casting, forexample, by admixing the ingredients described herein with water tocreate an aqueous mixture, for example a solution with optionallydispersed solids, applying the mixture to a surface, and drying offwater to create a film. Similarly, other compositions can be formed bydrying the mixture while it is confined in a desired shape.

In one contemplated class of embodiments, the water-soluble film isformed by casting a water-soluble mixture wherein the water-solublemixture is prepared according to the steps of:

-   (a) providing a mixture of water-soluble resin, water, and any    optional additives excluding plasticizers;-   (b) boiling the mixture for 30 minutes;-   (c) degassing the mixture in an oven at a temperature of at least    40° C.; optionally in a range of 40° C. to 70° C., e.g., about 65°    C.;-   (d) adding one or more enzymes, plasticizer, and additional water to    the mixture at a temperature of 65° C. or less; and-   (e) stirring the mixture without vortex until the mixture appears    substantially uniform in color and consistency; optionally for a    time period in a range of 30 minutes to 90 minutes, optionally at    least 1 hour; and-   (f) casting the mixture promptly after the time period of stirring    (e.g., within 4 hours, or 2 hours, or 1 hour).

If the enzyme is added to the mixture too early, e.g., with thesecondary additives or resin, the activity of the enzyme may decrease.Without intending to be bound by any particular theory, it is believedthat boiling of the mixture with the enzyme leads to the enzymedenaturing and storing in solution for extended periods of time alsoleads to a reduction in enzyme activity.

In one class of embodiments, high enzyme activity is maintained in thewater-soluble films according to the present disclosure by drying thefilms quickly under moderate to mild conditions. As used herein, dryingquickly refers to a drying time of less than 24 hours, optionally lessthan 12 hours, optionally less than 8 hours, optionally less than 2hours, optionally less than 1 hour, optionally less than 45 minutes,optionally less than 30 minutes, optionally less than 20 minutes,optionally less than 10 minutes, for example in a range of about 6minutes to about 10 minutes, or 8 minutes. As used herein, moderate tomild conditions refer to drying temperatures of less than 170° F. (77°C.), optionally in a range of about 150° F. to about 170° F. (about 66°C. to about 77° C.), e.g., 165° F. (74° C.). As the drying temperatureincreases, the enzymes tend to denature faster, whereas as the dryingtemperature decreases, the drying time increases, thus exposing theenzymes to solution for an extended period of time.

The film is useful for creating a packet to contain a composition, forexample laundry or dishwashing compositions, thereby forming a pouch.The film described herein can also be used to make a packet with two ormore compartments made of the same film or in combination with films ofother polymeric materials. Additional films can, for example, beobtained by casting, blow-molding, extrusion or blown extrusion of thesame or a different polymeric material, as known in the art. In one typeof embodiment, the polymers, copolymers or derivatives thereof suitablefor use as the additional film are selected from polyvinyl alcohols,polyvinyl pyrrolidone, polyalkylene oxides, polyacrylic acid, cellulose,cellulose ethers, cellulose esters, cellulose amides, polyvinylacetates, polycarboxylic acids and salts, polyaminoacids or peptides,polyamides, polyacrylamide, copolymers of maleic/acrylic acids,polysaccharides including starch and gelatin, natural gums such asxanthan, and carrageenans. For example, polymers can be selected frompolyacrylates and water-soluble acrylate copolymers, methylcellulose,carboxymethylcellulose sodium, dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, maltodextrin,polymethacrylates, and combinations thereof, or selected from polyvinylalcohols, polyvinyl alcohol copolymers and hydroxypropyl methylcellulose (HPMC), and combinations thereof.

The pouches and/or packets of the present disclosure comprise at leastone sealed compartment. Thus the pouches may comprise a singlecompartment or multiple compartments. The pouches may have regions withand without enzymes. In embodiments including multiple compartments,each compartment may contain identical and/or different compositions. Inturn, the compositions may take any suitable form including, but notlimited to liquid, solid and combinations thereof (e.g., a solidsuspended in a liquid). In some embodiments, the pouches comprises afirst, second and third compartment, each of which respectively containsa different first, second and third composition. In some embodiments,the compositions may be visually distinct as described in EP 2258820.

The compartments of multi-compartment pouches and/or packets may be ofthe same or different size(s) and/or volume(s). The compartments of thepresent multi-compartment pouches can be separate or conjoined in anysuitable manner. In some embodiments, the second and/or third and/orsubsequent compartments are superimposed on the first compartment. Inone embodiment, the third compartment may be superimposed on the secondcompartment, which is in turn superimposed on the first compartment in asandwich configuration. Alternatively the second and third compartmentsmay be superimposed on the first compartment. However it is also equallyenvisaged that the first, second and optionally third and subsequentcompartments may be attached to one another in a side by siderelationship. The compartments may be packed in a string, eachcompartment being individually separable by a perforation line. Henceeach compartment may be individually torn-off from the remainder of thestring by the end-user.

In some embodiments, multi-compartment pouches and/or packets includethree compartments consisting of a large first compartment and twosmaller compartments. The second and third smaller compartments aresuperimposed on the first larger compartment. The size and geometry ofthe compartments are chosen such that this arrangement is achievable.The geometry of the compartments may be the same or different. In someembodiments the second and optionally third compartment each has adifferent geometry and shape as compared to the first compartment. Inthese embodiments, the second and optionally third compartments arearranged in a design on the first compartment. The design may bedecorative, educative, or illustrative, for example to illustrate aconcept or instruction, and/or used to indicate origin of the product.In some embodiments, the first compartment is the largest compartmenthaving two large faces sealed around the perimeter, and the secondcompartment is smaller covering less than about 75%, or less than about50% of the surface area of one face of the first compartment. Inembodiments in which there is a third compartment, the aforementionedstructure may be the same but the second and third compartments coverless than about 60%, or less than about 50%, or less than about 45% ofthe surface area of one face of the first compartment.

The pouches and/or packets of the present disclosure may comprise one ormore different films. For example, in single compartment embodiments,the packet may be made from one wall that is folded onto itself andsealed at the edges, or alternatively, two walls that are sealedtogether at the edges. In multiple compartment embodiments, the packetmay be made from one or more films such that any given packetcompartment may comprise walls made from a single film or multiple filmshaving differing compositions. In one embodiment, a multi-compartmentpouch comprises at least three walls: an outer upper wall; an outerlower wall; and a partitioning wall. The outer upper wall and the outerlower wall are generally opposing and form the exterior of the pouch.The partitioning wall is interior to the pouch and is secured to thegenerally opposing outer walls along a seal line. The partitioning wallseparates the interior of the multi-compartment pouch into at least afirst compartment and a second compartment. In one class of embodiments,the partitioning wall may be the only enzyme containing film therebyminimizing the exposure of the consumer to the enzymes.

Pouches and packets may be made using any suitable equipment and method.For example, single compartment pouches may be made using vertical formfilling, horizontal form filling, or rotary drum filling techniquescommonly known in the art. Such processes may be either continuous orintermittent. The film may be dampened, and/or heated to increase themalleability thereof. The method may also involve the use of a vacuum todraw the film into a suitable mold. The vacuum drawing the film into themold can be applied for about 0.2 to about 5 seconds, or about 0.3 toabout 3, or about 0.5 to about 1.5 seconds, once the film is on thehorizontal portion of the surface. This vacuum can be such that itprovides an under-pressure in a range of 10 mbar to 1000 mbar, or in arange of 100 mbar to 600 mbar, for example.

The molds, in which packets may be made, can have any shape, length,width and depth, depending on the required dimensions of the pouches.The molds may also vary in size and shape from one to another, ifdesirable. For example, the volume of the final pouches may be about 5ml to about 300 ml, or about 10 to 150 ml, or about 20 to about 100 ml,and that the mold sizes are adjusted accordingly.

In one embodiment, the packet includes a first and a second sealedcompartment. The second compartment is in a generally superposedrelationship with the first sealed compartment such that the secondsealed compartment and the first sealed compartment share a partitioningwall interior to the pouch.

In one embodiment, the packet including a first and a second compartmentfurther includes a third sealed compartment. The third sealedcompartment is in a generally superposed relationship with the firstsealed compartment such that the third sealed compartment and the firstsealed compartment share a partitioning wall interior to the pouch.

In various embodiments, the first composition and the second compositionare selected from one of the following combinations: liquid, liquid;liquid, powder; powder, powder; and powder, liquid.

In various embodiments, the first, second and third compositions areselected from one of the following combinations: solid, liquid, liquidand liquid, liquid, liquid.

In one embodiment, the single compartment or plurality of sealedcompartments contains a composition. The plurality of compartments mayeach contain the same or a different composition. The composition isselected from a liquid, solid or combination thereof.

Heat can be applied to the film in the process commonly known asthermoforming. The heat may be applied using any suitable means. Forexample, the film may be heated directly by passing it under a heatingelement or through hot air, prior to feeding it onto a surface or onceon a surface. Alternatively, it may be heated indirectly, for example byheating the surface or applying a hot item onto the film. The film canbe heated using an infrared light. The film may be heated to atemperature of at least 50° C., for example about 50 to about 150° C.,about 50 to about 120° C., about 60 to about 130° C., about 70 to about120° C., or about 60 to about 90° C.

Alternatively, the film can be wetted by any suitable means, for exampledirectly by spraying a wetting agent (including water, a solution of thefilm composition, a plasticizer for the film composition, or anycombination of the foregoing) onto the film, prior to feeding it ontothe surface or once on the surface, or indirectly by wetting the surfaceor by applying a wet item onto the film.

Once a film has been heated and/or wetted, it may be drawn into anappropriate mold, preferably using a vacuum. The film can bethermoformed with a draw ratio of at least about 1.5, for example, andoptionally up to a draw ratio of 2, for example. The filling of themolded film can be accomplished by utilizing any suitable means. In someembodiments, the most preferred method will depend on the product formand required speed of filling. In some embodiments, the molded film isfilled by in-line filling techniques. The filled, open packets are thenclosed forming the pouches, using a second film, by any suitable method.This may be accomplished while in horizontal position and in continuous,constant motion. The closing may be accomplished by continuously feedinga second film, preferably water-soluble film, over and onto the openpackets and then preferably sealing the first and second film together,typically in the area between the molds and thus between the packets.

Any suitable method of sealing the packet and/or the individualcompartments thereof may be utilized. Non-limiting examples of suchmeans include heat sealing, solvent welding, solvent or wet sealing, andcombinations thereof. The water-soluble packet and/or the individualcompartments thereof can be heat sealed at a temperature of at least200° F. (93° C.), for example in a range of about 220° F. (about 105°C.) to about 290° F. (about 145° C.), or about 230° F. (about 110° C.)to about 280° F. (about 140° C.). Typically, only the area which is toform the seal is treated with heat or solvent. The heat or solvent canbe applied by any method, typically on the closing material, andtypically only on the areas which are to form the seal. If solvent orwet sealing or welding is used, it may be preferred that heat is alsoapplied. Preferred wet or solvent sealing/welding methods includeselectively applying solvent onto the area between the molds, or on theclosing material, by for example, spraying or printing this onto theseareas, and then applying pressure onto these areas, to form the seal.Sealing rolls and belts as described above (optionally also providingheat) can be used, for example.

The formed pouches may then be cut by a cutting device. Cutting can beaccomplished using any known method. It may be preferred that thecutting is also done in continuous manner, and preferably with constantspeed and preferably while in horizontal position. The cutting devicecan, for example, be a sharp item, or a hot item, or a laser, whereby inthe latter cases, the hot item or laser ‘burns’ through the film/sealingarea.

The different compartments of a multi-compartment pouches may be madetogether in a side-by-side style wherein the resulting, cojoined pouchesmay or may not be separated by cutting. Alternatively, the compartmentscan be made separately.

In some embodiments, pouches may be made according to a processincluding the steps of:

-   a) forming a first compartment (as described above);-   b) forming a recess within some or all of the closed compartment    formed in step (a), to generate a second molded compartment    superposed above the first compartment;-   c) filling and closing the second compartments by means of a third    film;-   d) sealing the first, second and third films; and-   e) cutting the films to produce a multi-compartment pouch.

The recess formed in step (b) may be achieved by applying a vacuum tothe compartment prepared in step (a).

In some embodiments, second, and/or third compartment(s) can be made ina separate step and then combined with the first compartment asdescribed in EP 2088187 or WO 2009/152031.

In other embodiments, pouches may be made according to a processincluding the steps of:

-   a) forming a first compartment, optionally using heat and/or vacuum,    using a first film on a first forming machine;-   b) filling the first compartment with a first composition;-   c) on a second forming machine, deforming a second film, optionally    using heat and vacuum, to make a second and optionally third molded    compartment;-   d) filling the second and optionally third compartments;-   e) sealing the second and optionally third compartment using a third    film;-   f) placing the sealed second and optionally third compartments onto    the first compartment;-   g) sealing the first, second and optionally third compartments; and-   h) cutting the films to produce a multi-compartment pouch.

The first and second forming machines may be selected based on theirsuitability to perform the above process. In some embodiments, the firstforming machine is preferably a horizontal forming machine, and thesecond forming machine is preferably a rotary drum forming machine,preferably located above the first forming machine.

It should be understood that by the use of appropriate feed stations, itmay be possible to manufacture multi-compartment pouches incorporating anumber of different or distinctive compositions and/or different ordistinctive liquid, gel or paste compositions.

Processes of Making the Compositions

The compositions of the present invention can be formulated into anysuitable form and prepared by any process chosen by the formulator,non-limiting examples of which are described in Applicants' examples andin U.S. Pat. No. 4,990,280; US20030087791A1; US20030087790A1;US20050003983A1; US20040048764A1; U.S. Pat. No. 4,762,636; U.S. Pat. No.6,291,412; US20050227891A1; EP1070115A2; U.S. Pat. No. 5,879,584; U.S.Pat. No. 5,691,297; U.S. Pat. No. 5,574,005; U.S. Pat. No. 5,569,645;U.S. Pat. No. 5,565,422; U.S. Pat. No. 5,516,448; U.S. Pat. No.5,489,392; U.S. Pat. No. 5,486,303 all of which are incorporated hereinby reference. The compositions of the invention or prepared according tothe invention comprise cleaning and/or treatment composition including,but not limited to, compositions for treating fabrics, hard surfaces andany other surfaces in the area of fabric and home care, including: aircare including air fresheners and scent delivery systems, car care,dishwashing, fabric conditioning (including softening and/orfreshening), laundry detergency, laundry and rinse additive and/or care,hard surface cleaning and/or treatment including floor and toilet bowlcleaners, granular or powder-form all-purpose or “heavy-duty” washingagents, especially cleaning detergents; liquid, gel or paste-formall-purpose washing agents, especially the so-called heavy-duty liquidtypes; liquid fine-fabric detergents; hand dishwashing agents or lightduty dishwashing agents, especially those of the high-foaming type;machine dishwashing agents, including the various tablet, granular,liquid and rinse-aid types for household and institutional use: car orcarpet shampoos, bathroom cleaners including toilet bowl cleaners; aswell as cleaning auxiliaries such as bleach additives and “stain-stick”or pre-treat types, substrate-laden compositions such as dryer addedsheets. Preferred are compositions and methods for cleaning and/ortreating textiles and/or hard surfaces, most preferably textiles. Thecompositions are preferably compositions used in a pre-treatment step ormain wash step of a washing process, most preferably for use in textilewashing step.

As used herein, the term “fabric and/or hard surface cleaning and/ortreatment composition” is a subset of cleaning and treatmentcompositions that includes, unless otherwise indicated, granular orpowder-form all-purpose or “heavy-duty” washing agents, especiallycleaning detergents; liquid, gel or paste-form all-purpose washingagents, especially the so-called heavy-duty liquid types; liquidfine-fabric detergents; hand dishwashing agents or light dutydishwashing agents, especially those of the high-foaming type; machinedishwashing agents, including the various tablet, granular, liquid andrinse-aid types for household and institutional use; liquid cleaning anddisinfecting agents, car or carpet shampoos, bathroom cleaners includingtoilet bowl cleaners; fabric conditioning compositions includingsoftening and/or freshening that may be in liquid, solid and/or dryersheet form; as well as cleaning auxiliaries such as bleach additives and“stain-stick” or pre-treat types, substrate-laden compositions such asdryer added sheets. All of such compositions which are applicable may bein standard, concentrated or even highly concentrated form even to theextent that such compositions may in certain aspect be non-aqueous.

Method of Use

The present invention includes a method for cleaning any surfaceincluding treating a textile or a hard surface or other surfaces in thefield of fabric and/or home care. In one aspect of the invention, themethod comprises the step of contacting the surface to be treated in apre-treatment step or main wash step of a washing process, mostpreferably for use in a textile washing step or alternatively for use indishwashing including both manual as well as automated/mechanicaldishwashing. In one embodiment of the invention the lipase variant andother components are added sequentially into the method for cleaningand/or treating the surface. Alternatively, the lipase variant and othercomponents are added simultaneously.

As used herein, washing includes but is not limited to, scrubbing, andmechanical agitation. Washing may be conducted with a foam compositionas described in WO08/101958 and/or by applying alternating pressure(pressure/vaccum) as an addition or as an alternative to scrubbing andmechanical agitation. Drying of such surfaces or fabrics may beaccomplished by any one of the common means employed either in domesticor industrial settings. The cleaning compositions of the presentinvention are ideally suited for use in laundry as well as dishwashingapplications. Accordingly, the present invention includes a method forcleaning an object including but not limiting to fabric, tableware,cutlery and kitchenware. The method comprises the steps of contactingthe object to be cleaned with a said cleaning composition comprising atleast one embodiment of Applicants' cleaning composition, cleaningadditive or mixture thereof. The fabric may comprise most any fabriccapable of being laundered in normal consumer or institutional useconditions. The solution may have a pH from 8 to 10.5. The compositionsmay be employed at concentrations from 500 to 15.000 ppm in solution.The water temperatures typically range from 5° C. to 90° C. The water tofabric ratio is typically from 1:1 to 30:1.

In one aspect the invention relates to a method of using the polypeptidewith at least 75% identity to SEQ ID NO: 3 for producing a composition.In one aspect the invention relates to use of the composition forcleaning an object.

In one aspect the invention relates to a method of producing acomposition comprising adding the variant. In one aspect the inventionrelates to a method of producing the composition, comprising adding apolypeptide with at least 75% identity to SEQ ID NO: 3, and asurfactant. In one aspect the invention relates to a method for cleaninga surface, comprising contacting a lipid stain present on the surface tobe cleaned with the cleaning composition. In one aspect the inventionrelates to a method for hydrolyzing a lipid present in a soil and/or astain on a surface, comprising contacting the soil and/or the stain withthe cleaning composition. In one aspect the invention relates to amethod of hydrolyzing a lipase substrate comprising contacting saidsubstrate with the variant.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

EXAMPLES Example 1 Assay Protocols

A stock of 6 mM 4-nitrophenyl palmitate (pNP-Palmitate) substratesolution was prepared by dissolving 226.5 mg of 4-nitrophenyl palmitate(Sigma cat. No. N2752) in 100 mL of absolute Ethanol (Merck cat. No.1.00983.0511)

A 625 uM pNP-Palmitate working solution was prepared by adding 10 mL ofstock substrate solution to 90 mL assay buffer (50 mL 1M TRIS pH 8.0(Sigma cat. No. T6066); 1.35 g Deoxycholate Solid/Sodium deoxycholateMonohydrate (Sigma cat. No. D5670); 0.7 g AOS (BIO-TERGE AS-40); up to500 mL Milli Q water).

The substrate and working solutions were stored in the dark at 4° C.until use.

TABLE 1 Model detergent ingredients Ingredient Amount (wt %) Linearalkylbenzenesulfonic acid (LAS) (97%) 5.00 Alcohol Ether sulfate (AEOS)(70.5%) 10.00 Sodium alkyl sulfate (AS) (90%) 4.50 Coco fatty acid(>99%) 1.00 AEO; alcohol ethoxylate with 7 mol EO (~100%) 5.00 MEA,monoethanol amine (99.5%) 0.30 MPG (>98%) 3.00 EtOH, propan-2-ol (90%)1.35 DTPA; diethylenetriaminepentaacetic acid, 0.10 pentasodium salt(40%) Na-citrate (100%) 4.00 Sodium formate (>95%) 1.00 NaOH, pellets(>99%) 0.66 Water up to 100

A: Thermostability Assay

Thermostability was determined by measuring the enzymatic activitypresent in culture supernatants of variants or wild-type controlsexposed to 50° C.

Heat treatment of 10 uL supernatant samples in 96-well Abgene PCR plates(Thermo Scientific cat. No. AB0800) were conducted in a programmablethermal cycler (T-ROBOT) for 10 minutes at room temperature (control) or50° C. and subsequently cooled down to 4° C. The samples were diluted120 times with assay dilution buffer (250 mL 1M TRIS pH 8.0 (Sigma cat.No. T6066); 45 mL 1M CaCl₂ (Sigma cat. No. C5080); 3.75 mL 30% BRIJ(Sigma cat. No. B4184); up to 5000 mL Milli Q water).

Reaction mixtures were prepared in 384-well plate (Nunc cat. No. 262160)by adding 10 uL of diluted and heat treated supernatant sample to 40 uLof 625 uM pNP-Palmitate working solution. Enzymatic activity wasmeasured at 25° C. by monitoring the rate of increase in absorbance at405 nm using a Infinite M1000 reader (TECAN, Switzerland).

The residual activity (RA) for variants and wildtype controls werecalculated as the percentage of enzymatic activity remaining afterincubation at 50° C. relative to enzymatic activity remaining afterincubation at room temperature.

The variants with higher thermal stability were picked with respect tothe wild types grown in the plates with IF of >1.2 .

B: Thermostability in Detergent Assay

Stability in detergent was determined by measuring the enzymaticactivity present in culture supernatants of variants or wild-typecontrols after exposure to detergent at 30° C.

Detergent treatment was conducted by adding 20 uL supernatant and 80 uLof a 30% Model detergent diluted in assay dilution buffer into wells of96-well microtitre plates (Nunc cat. No. 269620) which were shaked for 5minutes at 1000rpm. Two identical plates were produced whereof one platewas incubated for 16 hours at 4° C. (unstressed plate) and the otherplate was incubated at 30° C. (stressed plate).

Reaction mixtures were prepared in 384-well plates (Nunc cat. No.262160). Supernatant samples from unstressed and stressed plates werediluted 5X with assay dilution buffer. 10 uL of diluted unstressed ordiluted tressed sample were added to 40 uL of 625 uM pNP-Palmitateworking solution. Enzymatic activity was measured at 25° C. bymonitoring the rate of increase in absorbance at 405 nm using anInfinite M1000 reader (TECAN, Switzerland).

The residual activity (RA) for variants and reference were calculated asthe percentage of enzymatic activity remaining after incubation at 50°C. relative to enzymatic activity remaining after incubation at 4° C.

The variants with higher thermal stability were picked with respect tothe wild types grown in the plates with IF of >1.2.

C: Detergent Stability Assay

Detergent stability at 4° C. was calculated by comparing unstressedsamples prepared according to Assay protocol A with unstressed samplesprepared accoding to Assay protocol B.

D: Performance Screening Assay

Performance i.e. hydrolytic activity was determined by measuring therelease of 4-methylumbelliferone (4-MU) in culture supernatants ofvariants and wild-type controls.

Cellulose (Avicel) was coated with a mixture of triglyceride and4-methylumbelliferyl oleate (4-MU oleate) in an initial substratepreparation step. In the assay, the coated cellulose fibers weresuspended in a buffer, a detergent solution, and a lipase solution. Thelipase activity monitored in the cellulose suspension by measuring thefluorescence of released 4-methylumbelliferone (4-MU) at Ex: 365 nm andEm: 445 nm as a function of time.

Chemicals:

-   4-methylumbelliferyl oleate (oleic acid 4-methylumbelliferyl ester,    Sigma Aldrich 75164)-   n-Hexane (Sigma-Aldrich 15671)-   Olive oil (Sigma-Aldrich O-1514)-   Cellulose cotton linters, Avicel H-101 (Sigma-Aldrich 11365) HEPES    (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid,    N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid,    Sigma-Aldrich H33759)-   Calcium chloride dihydrate-   Triton™ X-100 (4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene    glycol, t-Octyl phenoxypolyethoxyethanol, Polyethylene glycol    tert-octylphenyl etherSigma-Aldrich T9284)-   4 mol/L NaOH (Fluka 35274)-   Model Detergent

Coated cellulose fibers were prepared by pipetting 668 uL 4-MU-oleatestock solution (4mg/mL 4-Methylumbelliferyl oleate in hexane) and 3.03mL olive oil stock solution into 100 mL hexane in a round bottomed flaskfor rotational evaporator, and mixed properly before 10 g cellulosefibers was added. The flask was mounted on a rotational evaporator, andhexane was removed by evaporation at reduced pressure and constantmixing (by rotation) to ensure uniformly coating of the substrate on thecellulose. After complete removal of hexane, the coated cellulose fiberswere transferred to a brown flask and the material was stored protectedfrom light at −20° C. until use.

The assay was conducted by transferring 80 uL of Substrate suspension toeach well in a black micro-well microtiter plate (e.g. Nunclon surface,96 well black plates with black bottom, NUNC 137101). It is important tokeep a constant, thorough mixing of the Substrate suspension whenpipetting to ensure that the same amount of coated cellulose fibers aretransferred to each well of the microtiter plate. 10 uL of the Detergentworking solution was added to the wells.

The assay was started with addition of 10 uL of culture supernatants ofvariants or wild-type controls. The enzyme (supernatant) should be addedimmediately after addition of the Detergent working solution (1.65gModel J Detergent (DC-2012-0057-6); adjust to 50 mL with Milli Q water).For the blank, 10 uL Lipase dilution buffer (200 mM HEPES; 1mM CaCl2; 10ppm Triton X-100; adjusted to pH 8.3 with 4M NaOH) was added.

The enzyme activity was followed by measuring the fluorescence (kineticmode) every 30 seconds at Ex: 365 nm and Em: 445 nm from 1 to 6 minutesafter enzyme addition. The plate was mixed by shaking for 5 secondsbefore first measurement but not between the following measurements. Theassay was run at room temperature, i.e. approximately at 23° C.

The activity was calculated as the slope of a plot of fluorescenceversus time (units: RFS/minute, where RFS is the Relative FlourescenceSignal) using the 1-6 minute time window.

Improvement Factor

The improvement factor (IF) correlates the performance of a variantenzyme with that of the reference enzyme at the same proteinconcentration. Calculation of the improvement factor based on ResidualActivity (RA) were done according to the following equaition: IF=(RA ofthe variant)/(RA of the Wildtype). Calculation of the improvement factorbased on RFS/minute were done according to the following equaition:IF=(RFS of the variant)/(RFS of the Wildtype).

An improvement factor that is greater than 1 (IF>1) indicates animproved performance by a variant as compared to the reference, while aIF of 1 (IF=1) identifies a variant that performes the same as thereference, and an IF of less than 1 (IF<1) identifies a variant thatperforms worse that the reference.

Example 2 Generation of Rhizomucor miehei Lipase Site-SaturationLibraries

The gene of Rhizomucor miehei lipase (RmL) SEQ ID No: 1 was cloned intothe expression vector, pBGMH0016 and transformed in the expression host,Aspergillus oryzae COIs1300.

SEQ ID No: 1 sets forth the coding nucleotide sequence of the RmLincluding the sequence (underligned) cleaved of:

atg gtt ctc aag cag cgt gca aac tac cta gga tttctg att gta ttc ttc acg gcc ttc ctg gtg gaa gcggta ccc atc aag aga caa tcg aat tcc acg gtc gacagt ctg ccg cct gcc tcg gtc ctc atc ccc tcg agaacc tcg gca cct tca tca tca cca agc aca acc gaccct gaa gct cca gcc atg agt cgc aat gga ccg ctgccc tcg gat gta gag act aaa tat ggc atg gct ttgaat gct act tcc tat ccg gat tct gtg gtc caa gca atg agc att gat ggt ggtatc cgc gct gcg acc tcg caa gaa atc aat gaa ttg act tat tac act aca ctatct gcc aac tcg tac tgc cgc act gtc att cct gga gct acc tgg gac tgt atccac tgt gat gca acg gag gat ctc aag att atc aag act tgg agc acg ctc atctat gat aca aat gca atg gtt gca cgt ggt gac agc gaa aaa act atc tat atcgtt ttc cga ggt tcg agc tct atc cgc aac tgg att gct gat ctc acc ttt gtgcca gtt tca tat cct ccg gtc agt ggt aca aaa gta cac aag gga ttc ctg gacagt tac ggg gaa gtt caa aac gag ctt gtt gct act gtt ctt gat caa ttc aagcaa tat cca agc tac aag gtt gct gtt aca ggt cac tca ctc ggt ggt gct actgcg ttg ctt tgc gcc ctg gat ctc tat caa cga gaa gaa gga ctc tca tcc agcaac ttg ttc ctt tac act caa ggt caa cca cgg gta ggc gac cct gcc ttt gccaac tac gtt gtt agc acc ggc att cct tac agg cgc acg gtc aat gaa cga gatatc gtt cct cat ctt cca cct gct gct ttt ggt ttt ctc cac gct ggc gag gagtat tgg att act gac aat agc cca gag act gtt cag gtc tgc aca agc gat ctggaa acc tct gat tgc tct aac agc att gtt ccc ttc aca agt gtt ctt gac catctc tcg tac ttt Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp His Leu SerTyr Phe ggt atc aac aca ggc ctc tgt act

SEQ ID No: 2 sets forth the coding amino acid sequence of the RmLincluding the sequence (underligned) cleaved of.

MVLKQRANYL GFLIVFFTAF LVEAVPIKRQ SNSTVDSLPPASVLIPSRTS APSSSPSTTD PEAPAMSRNG PLPSDVETKY GMALNATSYP DSVVQAMSIDGGIRAATSQE INELTYYTTL SANSYCRTVI PGATVVDCIHC DATEDLKIIK TWSTLIYDTNAMVARGDSEK TIYIVFRGSS SIRNWIADLT FVPVSYPPVS GTKVHKGFLD SYGEVQNELVATVLDQFKQY PSYKVAVTGH SLGGATALLC ALDLYQREEG LSSSNLFLYT QGQPRVGDPAFANYVVSTGI PYRRTVNERD IVPHLPPAAF GFLHAGEEYW ITDNSPETVQ VCTSDLETSDCSNSIVPFTS VLDHLSYFGI NTGLCT

SEQ ID No: 3 sets forth the amino acid sequence of the RmL matureprotein:

SIDGGIRAAT SQEINELTYY TTLSANSYCR TVIPGATVVDC IHCDATEDLK IIKTWSTLIYDTNAMVARGD SEKTIYIVFR GSSSIRNWIA DLTFVPVSYP PVSGTKVHKG FLDSYGEVQNELVATVLDQF KQYPSYKVAV TGHSLGGATA LLCALDLYQR EEGLSSSNLF LYTQGQPRVGDPAFANYVVS TGIPYRRTVN ERDIVPHLPP AAFGFLHAGE EYWITDNSPE TVQVCTSDLETSDCSNSIVP FTSVLDHLSY FGINTGLCT

Site-saturation libraries (SSL) were generated using Splicing by OverlapExtension PCR (SOE PCR) in each mentioned position in the RmL gene.Three PCR reactions were involved: 1) Generation of N-terminal fragmentwith the flanking N-terminal forward primer and the reverse mutagenicprimer; 2) Generation of C-terminal fragment with flanking C-terminalreverse primer and mutagenic forward primer which had a region ofcomplementarity to the reverse mutagenic primer; and 3) SOE PCR withDpnI digested and purified N- and C-fragments. The polymerase used forthe PCR reaction was Phusion DNA polymerase (Finnzymes, Cat. No.: F530L.DpnI was from New England Biolabs (Cat. No.: R0176S).

The SOE PCR product was purified and transformed in to the Aspergillushost, where site-specific homologous recombination in the Aspergilluschromosome took place.

Colonies picked from the transformed plate were inoculated in 96-wellculture plates (Nunc cat. No. 260887) containing M400 media (50 g/LMaltodextrin; 2 g/L Magnesium sulfate; 2 g/L Potassium dihydrogenphosphate; 4 g/L Citric acid monohydrous; 8 g/L Yeast Extract; 2 g/LUrea; 0.5 ml/L tracemetal (KU6) solution; and 0.5 g/L Calcium chloride)and grown at 34° C. for 4 days. Supernatants were recovered from theplates by centrifugation and were used for all the assays.

The position and mutation of the SSL variants were verified by standardsequencing.

Example 3 Improved Variants

TABLE 2 Results from testing the variants in four different assaysSubstitution Assay A (IF) Assay B (IF) Assay C (IF) Assay D (IF) S1C 1.21.1 S1F 1.4 1.1 S1G 1.3 1.0 S1H 1.2 1.0 S1I 1.3 1.2 S1L 1.3 S1M 1.5 1.21.2 S1P 1.5 S1Q 1.3 1.2 S1R 1.5 1.5 S1V 1.3 1.0 S1W 1.2 1.0 S1Y 1.5 1.2I2A 1.3 1.0 I2C 1.7 1.3 I2D 1.1 1.2 I2M 1.2 I2N 1.4 1.4 I2P 1.4 1.5 I2Q1.2 1.5 1.0 I2R 1.4 1.0 I2S 1.3 1.0 I2T 1.6 1.5 1.7 I2V 1.5 1.1 D3A 1.3D3E 1.1 1.2 1.2 D3G 1.4 1.0 D3K 1.3 1.0 D3Q 1.0 D3S 1.0 D3T 1.0 D3V 1.3G4A 1.0 1.0 1.0 G4P 1.0 1.1 1.2 G4Q 1.5 1.1 G4R 1.2 1.1 G4S 1.0 1.3 1.0G4T 1.3 1.3 G5E 1.3 G5K 1.3 1.3 G5S 1.1 1.2 G5T 1.1 1.0 G5V 1.2 G5Y 1.01.1 1.0 I6A 1.4 I6H 1.5 1.4 I6K 1.4 1.2 1.6 1.1 I6N 1.4 1.4 I6P 1.3 1.11.3 I6Q 1.4 1.3 I6R 1.3 1.3 I6S 1.2 I6V 1.3 1.4 I6W 1.5 R7P 1.0 1.1 A8C1.4 1.4 A8G 1.0 A8H 1.1 A8N 1.2 A8P 1.3 1.2 A8Q 1.1 A8R 1.7 1.2 A8S 1.41.4 A8T 1.5 1.0 A8W 1.6 1.2 A8Y 1.1 A9Y 1.3 T10G 1.3 T10R 1.3 T10S 1.11.3 1.0 T10Y 1.0 S11G 2.0 S11H 1.3 1.6 S11I 1.2 1.8 S11L 1.4 1.4 S11P1.3 1.3 1.5 S11Q 1.3 S11R 1.4 1.2 2.1 S11T 1.2 Q12A 1.4 Q12I 1.4 1.2Q12K 1.4 Q12L 1.2 1.5 Q12M 1.6 1.5 Q12N 1.5 Q12R 1.6 1.6 Q12S 1.2 1.2Q12T 1.3 1.5 E16P 1.4 E16R 1.3 E16S 1.4 E16T 1.4 1.2 E16W 1.2 Y19C 1.31.6 Y19E 1.3 Y19H 1.2 Y19L 1.6 Y19W 1.3 Y19T 1.9 R30A 1.2 1.7 R30F 1.21.6 R30H 1.3 R30I 1.5 R30L 1.4 1.2 1.7 1.0 R30N 1.4 R30P 1.3 1.3 1.8R30S 1.4 1.3 R30T 1.2 1.8 R30V 1.5 R30W 1.3 T31A 1.2 T31C 1.4 T31D 1.3T31E 1.2 1.0 T31F 1.4 T31G 1.4 1.5 T31H 1.3 T31I 1.2 T31K 1.5 T31P 1.4T31Q 1.4 1.3 T31R 1.2 T31S 1.4 T31V 1.2 1.4 P34A 1.6 P34G 2.1 P34V 1.6A36D 1.5 A36E 1.2 1.8 A36G 1.2 1.8 A36K 1.2 1.2 A36Q 1.2 1.0 A36R 1.2A36S 1.3 A36V 1.2 T37A 1.3 2.1 T37E 1.5 T37G 1.5 T37S 1.5 T37V 1.5 D39F1.7 D39K 1.4 D39L 1.5 D39N 1.2 D39S 1.4 C40A 1.4 2.0 C40D 1.2 C40E 2.8C40G 1.4 1.7 C40I 2.4 C40L 1.6 C40R 1.7 C40V 1.8 1.4 I41A 1.3 2.3 I41D1.4 I41E 1.3 I41G 1.3 I41H 1.3 1.6 I41K 1.5 I41L 1.4 1.5 I41P 1.3 2.2I41Q 1.3 I41R 1.4 1.5 I41S 1.4 I41T 1.3 I41V 1.3 1.7 I41W 1.3 H42A 1.31.3 H42E 1.2 H42G 1.2 1.3 H42K 1.1 1.1 H42L 1.3 1.3 1.1 H42M 1.3 H42R1.3 1.2 1.3 1.1 H42S 1.3 H42T 1.3 H42V 1.2 1.0 1.3 D44A 1.3 1.3 1.3 D44C1.2 D44E 1.3 D44G 1.5 1.3 1.3 1.1 D44H 1.3 1.2 D44K 1.3 1.2 1.3 D44M 1.4D44P 1.4 1.2 1.5 D44Q 1.4 D44R 1.3 D44S 1.4 1.1 D44V 1.4 1.2 D44W 1.3I51A 1.2 1.3 I51D 1.9 I51G 1.2 1.9 I51H 1.2 I51K 1.2 I51L 1.4 I51M 1.2I51V 1.4 1.3 1.8 I51Y 1.8 I52L 1.3 1.5 I52V 1.3 1.7 K53A 1.3 K53D 1.3K53G 1.4 1.8 K53H 1.2 1.6 K53I 1.4 K53L 1.2 1.6 K53M 1.2 K53P 2.5 K53Q1.4 1.8 K53R 1.4 2.3 K53S 1.3 K53V 1.4 T54A 1.3 1.0 T54D 1.5 T54E 1.4T54G 1.5 T54I 1.4 1.1 T54K 1.3 1.4 T54L 1.3 1.2 T54S 1.5 T54V 1.4 1.81.0 S56A 1.7 S56P 2.3 S56Q 1.4 1.4 S56R 1.7 S56T 1.4 2.4 S56V 1.7 S56W2.3 L58C 1.2 1.2 1.5 L58G 1.4 1.2 L58I 1.2 L58P 1.3 1.4 L58R 1.3 1.3L58S 1.4 L58V 1.5 L58W 1.3 1.2 I59A 1.3 I59D 1.1 1.8 I59F 1.2 I59G 1.5I59H 1.4 1.3 I59L 1.1 I59M 1.5 I59P 2.0 I59R 1.5 1.4 I59S 1.5 I59T 1.41.1 I59V 1.5 1.1 1.9 D61A 1.8 D61G 1.5 D61M 2.5 D61S 2.6 D70A 1.3 1.7D70E 1.2 D70G 1.2 1.5 D70R 1.4 S71E 1.3 S71K 1.2 1.4 S71N 1.5 1.4 S71Q1.4 S71R 1.2 1.2 S71W 1.2 2.0 1.1 S71Y 1.2 1.2 E72A 1.3 1.5 1.4 E72C 1.2E72D 1.5 E72G 1.2 1.7 E72K 1.5 1.5 1.0 E72R 1.3 1.4 E72S 1.3 E72T 1.32.2 K73E 1.5 1.3 1.4 K73G 1.6 1.4 1.4 K73L 1.2 K73N 1.2 1.3 K73Q 1.3 1.3K73R 1.3 1.2 1.3 K73S 1.2 K73T 1.3 S83A 1.4 S84A 1.3 S84E 1.3 S84L 1.3S84P 1.3 S84W 1.4 S84Y 1.4 R86A 2.3 R86G 1.3 1.8 R86I 1.3 R86K 1.3 R86M1.2 R86W 1.6 W88M 1.2 W88Q 1.2 W88R 1.4 1.3 A90D 1.2 A90F 1.3 1.5 A90G1.2 A90L 1.7 A90S 1.3 A90T 1.3 A90V 1.3 A90W 1.3 D91A 1.4 D91E 1.2 D91F1.4 D91G 1.3 D91L 1.4 D91Q 1.2 D91S 1.3 D91T 1.2 D91Y 1.3 1.3 L92A 1.41.3 L92C 1.4 L92D 1.5 L92F 1.4 1.0 L92P 1.7 L92S 1.4 1.3 2.0 L92T 1.51.2 L92V 1.3 1.2 1.4 L92W 2.0 T93A 1.3 1.1 1.7 1.1 T93I 1.1 T93R 1.4 1.3T93V 1.4 1.4 1.4 V95C 1.4 V95G 1.6 1.5 V95I 1.3 V95Q 1.2 V95R 1.5 1.5V95S 1.4 P96A 1.2 1.0 P96C 1.9 1.2 1.4 1.1 P96D 1.2 1.2 P96E 1.5 1.3P96G 1.5 1.2 1.5 P96N 1.3 1.4 P96Q 2.2 1.7 1.0 P96R 1.5 1.0 P96S 1.8 1.5P96W 2.0 1.2 1.1 P96Y 2.0 1.4 1.3 S98A 1.5 1.3 1.4 S98D 1.5 1.4 S98E 1.41.3 1.1 S98G 1.4 1.2 S98K 1.2 S98N 1.5 S98P 1.5 S98R 1.3 S98T 1.2 S98V1.4 1.4 S98W 1.3 P100A 1.3 1.7 P100E 1.4 P100G 1.5 P100K 1.3 P100L 1.3P100R 1.2 1.3 P100S 1.3 P100V 1.4 P100W 1.5 1.2 P101D 1.2 P101H 1.2P101R 1.3 P101S 1.3 P101V 1.2 V102H 1.6 V102I 1.2 V102P 1.3 V102R 1.22.0 S103A 1.4 1.3 1.4 S103C 1.3 S103D 1.2 1.3 S103F 1.3 S103G 1.6 1.31.4 S103L 1.2 1.4 S103P 1.5 2.1 S103R 1.2 1.4 2.0 S103V 1.2 1.7 G104A1.3 G104C 1.4 1.0 G104D 1.4 G104E 1.3 G104K 1.7 1.4 G104L 1.5 1.2 1.2G104M 1.2 1.5 1.0 G104P 1.2 1.0 G104Q 1.2 G104R 1.5 1.3 1.4 G104V 1.3G104W 1.5 G104Y 1.2 K106A 1.2 1.4 K106C 1.2 K106G 1.5 K106L 1.5 1.5K106P 1.3 1.7 K106Q 1.3 K106R 1.4 K106S 1.3 K106T 1.4 1.1 K106W 1.7K109A 1.2 1.3 K109D 1.3 K109E 1.2 1.5 K109G 1.4 K109H 1.4 K109L 1.3 1.3K109N 1.2 K109P 1.3 K109Q 1.4 K109R 1.7 1.4 1.9 1.1 K109S 1.2 1.3 1.0K109T 1.2 G110C 1.5 1.3 G110P 1.3 G110S 1.4 1.3 G110V 1.5 1.2 L112F 1.21.2 L112H 1.2 1.0 L112Q 1.3 1.4 1.4 L112R 1.3 1.3 1.1 L112W 1.5 1.2 1.0D113L 1.9 D113M 1.6 D113R 1.9 D113T 1.4 D113W 1.8 G116A 1.2 G116C 1.4G116D 1.5 G116F 1.4 G116R 1.4 G116S 1.2 G116T 1.3 G116V 1.3 E117A 1.41.3 E117D 1.5 E117G 1.8 E117N 1.4 E117P 1.4 E117R 1.3 1.3 E117S 1.3E117V 2.5 Q119D 1.3 1.0 Q119E 1.2 1.4 1.3 Q119F 1.4 Q119I 1.3 Q119R 1.31.6 1.4 1.0 Q119S 1.4 1.3 1.4 Q119T 1.4 1.4 Q119V 1.3 N120A 1.4 N120D1.5 N120E 1.2 N120G 1.3 N120H 1.4 N120L 1.3 N120M 1.3 1.2 N120S 1.5N120T 1.3 N120V 1.2 N120Y 1.2 A124C 1.4 A124D 1.2 A124G 1.3 1.4 A124H1.3 A124L 1.2 1.3 1.6 A124M 1.6 1.6 A124Q 1.4 A124R 1.5 1.6 A124S 1.31.3 1.3 A124V 1.2 1.3 A124W 1.3 1.3 T125A 1.5 1.2 2.0 T125D 1.3 2.2T125E 1.4 T125G 1.3 2.6 T125I 1.6 T125K 1.3 T125L 1.9 T125M 1.2 1.3 1.5T125Q 2.9 T125R 1.4 1.3 1.7 T125S 1.3 1.3 2.3 T125V 1.5 1.4 1.7 L127A1.2 L127F 1.3 1.3 L127G 1.2 L127H 1.2 1.2 L127I 1.3 L127Q 1.3 L127R 1.41.2 L127S 1.3 1.3 1.1 L127V 1.2 1.4 L127Y 1.2 D128A 1.2 1.0 1.5 D128E1.3 D128G 1.0 2.0 D128H 1.9 D128M 1.5 D128Q 1.3 1.0 D128S 1.6 D128T 1.21.1 K131A 1.3 1.4 1.3 K131G 1.6 1.2 1.3 K131I 1.2 1.1 K131M 1.5 1.3 1.6K131S 1.2 1.2 K131T 1.7 1.3 1.7 K131V 1.3 1.3 1.3 K131W 1.2 1.6 Q132G1.5 Q132S 1.8 Q132V 1.2 Y133A 1.3 1.0 Y133E 1.3 1.3 1.1 Y133K 1.2 Y133L1.5 Y133M 1.3 Y133R 1.4 Y133V 1.5 Y133W 1.4 1.9 1.1 P134A 1.3 1.4 1.3P134D 1.4 1.5 1.7 P134E 1.4 1.2 P134G 1.4 1.3 1.6 P134K 1.4 1.2 1.5P134R 1.4 1.5 1.7 P134S 1.3 1.2 P134T 1.4 1.4 P134V 1.3 S135A 1.2 1.5S135C 1.7 1.3 S135G 1.4 1.3 1.6 S135P 1.4 3.8 S135Q 1.4 S135R 1.3 1.9S135T 1.3 S135V 1.7 S135W 1.7 1.5 1.3 S135Y 1.3 1.3 1.8 K137A 1.5 K137F1.9 K137G 1.8 K137P 1.5 2.5 K137R 1.7 1.7 Y158A 1.3 Y158C 1.3 1.2 Y158G1.4 1.3 Y158I 1.3 1.2 1.0 Y158L 1.6 Y158N 1.2 1.2 Y158R 1.4 1.5 Y158S1.2 1.3 1.0 Y158T 1.2 Y158V 1.3 Y158W 1.3 Q159G 2.0 Q159T 1.5 R160A 1.31.4 R160D 1.2 R160G 1.2 R160I 1.2 R160L 1.4 R160Q 1.1 1.5 R160V 1.4 1.61.3 E161C 1.2 1.4 E161G 1.3 E161I 1.2 E161K 1.4 E161L 1.2 1.3 E161P 1.6E161Q 1.2 1.6 E161R 1.4 1.4 E161S 1.2 1.0 E162A 1.2 1.3 1.4 E162D 1.2E162G 1.2 1.4 1.0 E162L 1.6 1.2 1.2 E162M 1.3 1.2 E162P 1.2 1.2 1.5 1.3E162R 1.5 1.3 1.3 E162S 1.6 1.3 1.5 1.9 E162V 1.4 1.2 1.9 E162W 1.2 1.7G163A 1.2 1.3 1.0 G163D 1.2 1.2 1.1 G163E 1.3 1.4 1.3 1.1 G163H 1.4G163L 1.4 1.2 G163M 1.4 1.1 G163Q 1.4 1.3 1.2 G163V 1.5 G163Y 1.3 S165A1.2 1.3 S165C 1.3 S165G 1.3 1.2 1.0 S165K 1.2 1.2 S165L 1.4 1.5 S165M1.2 S165P 1.2 S165R 1.4 1.4 1.3 S165V 1.4 1.4 S165W 1.2 1.4 S166A 1.31.3 1.2 1.4 S166C 1.2 1.6 1.3 S166E 1.7 S166G 1.2 1.3 1.5 1.5 S166L 1.2S166P 1.2 1.3 S166R 1.4 S166W 1.4 1.1 S167C 1.2 S167F 1.3 S167H 1.3 1.7S167I 1.3 1.2 S167L 1.2 1.2 S167M 1.2 S167R 1.2 1.0 S167V 1.2 1.0 N168A1.2 1.3 N168C 1.3 N168D 1.2 N168G 1.2 N168R 1.4 N168S 1.2 N168V 1.4N168W 1.2 F170A 1.3 1.4 F170C 1.2 F170E 1.2 1.2 F170G 1.4 1.4 F170H 1.6F170I 1.4 F170K 1.4 F170L 1.6 1.2 F170M 1.2 F170Q 1.2 F170R 1.6 F170S1.5 F170V 1.3 1.2 F170W 1.2 F170Y 1.2 D181A 1.6 D181C 1.2 D181E 1.2D181G 1.5 1.2 D181L 1.3 D181Q 1.2 D181R 1.2 D181S 1.2 D181V 1.7 D181W1.4 1.5 P182A 2.1 1.3 P182C 1.7 1.3 P182F 1.2 2.4 P182G 1.6 1.6 P182I1.2 1.3 1.7 1.0 P182L 2.1 1.2 P182R 1.3 1.8 1.5 P182S 1.8 2.0 P182T 1.31.5 1.0 P182V 2.4 1.6 P182W 2.2 1.4 P182Y 1.3 1.3 1.2 A183E 1.5 A183V1.5 N186A 1.2 1.2 N186C 1.4 N186E 1.2 1.2 N186G 1.4 1.2 1.6 N186H 1.4N186K 1.2 N186L 1.2 N186R 1.4 1.3 N186S 1.2 1.4 1.4 N186T 1.3 N186W 1.3N186Y 1.2 1.3 V189A 1.2 V189C 1.6 V189G 1.3 1.3 V189L 1.3 1.3 V189M 1.5V189P 1.4 1.5 V189R 1.3 1.7 V189S 1.5 S190A 1.4 1.3 1.7 S190E 1.3 1.31.3 S190G 1.6 1.3 1.9 S190I 1.2 S190K 1.2 1.4 1.3 S190N 1.5 S190R 1.61.3 1.6 S190V 1.3 1.3 S190W 1.2 G192A 1.5 G192I 1.5 1.1 G192L 1.3 1.7G192M 1.2 1.4 G192P 1.3 1.7 G192R 1.2 1.5 G192T 1.3 1.5 1.3 G192V 1.21.6 P194A 1.3 1.7 P194D 1.3 P194E 1.3 1.2 P194F 1.4 P194G 1.3 1.6 P194H1.2 P194L 1.4 1.6 P194Q 1.2 P194T 1.4 P194V 1.6 R196A 1.3 R196Q 1.2R196Y 1.2 E201A 1.3 1.4 1.9 E201D 1.2 1.3 E201G 1.3 1.4 1.6 1.2 E201L1.2 1.5 1.1 E201P 1.2 E201S 1.2 E201T 1.2 E201V 1.3 1.7 R202I 1.2 R202M1.2 I204E 1.2 I204K 1.4 1.2 I204L 1.4 I204P 1.4 1.5 I204R 1.3 I204V 1.41.2 L208A 1.3 1.3 L208E 1.2 L208G 1.5 L208M 1.4 1.3 L208P 1.9 L208R 1.31.3 L208S 1.3 1.4 L208W 1.3 1.3 P210A 1.3 P210F 2.0 P210L 1.4 P210Q 1.7A211C 1.6 A211E 1.2 A211G 1.3 1.2 A211P 1.4 A211S 1.2 1.6 A211V 1.3 1.8A211W 1.3 1.5 A212D 1.5 1.5 1.5 A212E 1.3 1.3 A212G 1.5 1.3 1.4 A212H1.4 1.2 1.5 A212K 1.2 1.5 A212N 1.3 1.4 A212P 1.4 1.6 A212Q 1.2 1.4A212R 1.4 1.2 A212S 1.3 1.2 1.5 A212T 1.3 A212V 1.4 A212W 1.4 F213A 1.21.2 1.2 F213E 1.4 1.0 F213G 1.4 1.3 F213H 1.3 F213L 1.2 1.0 F213M 1.21.2 F213P 1.3 1.2 F213Q 1.4 F213R 1.2 1.2 F213V 1.3 1.3 1.3 F213W 1.4E220A 1.2 1.2 E220C 1.6 1.6 E220D 1.2 1.2 E220G 1.4 1.2 1.7 E220L 1.4E220P 1.2 1.3 E220R 1.7 1.2 E220S 1.7 1.3 1.6 E220T 1.6 E220W 1.3 1.5T225A 1.2 T225L 1.4 1.2 1.5 T225M 1.2 T225P 1.3 T225Q 1.5 T225S 1.2D226A 1.4 1.2 1.3 D226C 1.3 1.2 1.4 1.4 D226E 1.3 1.2 D226F 1.4 D226G1.3 1.5 1.3 1.2 D226P 1.2 D226R 1.4 1.3 D226S 1.6 D226T 1.2 D226V 1.31.7 D226W 1.2 1.2 1.7 D226Y 1.2 N227A 1.3 N227C 1.3 1.3 N227G 1.3 1.41.1 N227P 1.2 1.1 N227R 1.2 1.2 N227S 1.4 1.3 1.0 N227V 1.2 N227W 1.3S228A 1.2 1.3 S228C 1.7 S228D 1.4 S228G 1.2 1.7 S228L 1.2 1.4 S228P 1.3S228R 1.5 1.2 1.3 1.0 S228V 1.2 1.4 S228W 1.4 P229A 1.3 P229I 1.2 P229L1.4 1.2 1.5 P229N 1.3 1.2 P229R 1.3 1.0 P229S 1.4 1.2 1.0 P229T 1.2E230A 1.2 1.3 E230G 1.2 1.5 1.3 E230H 1.5 1.5 1.0 E230L 1.4 E230P 1.41.6 1.3 E230Q 1.2 E230R 1.2 1.6 1.4 E230S 1.4 2.0 1.7 E230T 1.5 E230V1.2 E230W 1.5 1.2 1.1 T231A 1.2 1.4 T231C 1.4 1.4 T231G 1.5 1.7 1.6T231H 1.2 1.2 1.4 T231K 1.4 1.3 T231R 1.5 1.3 1.6 1.0 T231S 1.3 1.4T231V 1.3 T231W 1.5 T231Y 1.5 1.8 Q233A 1.2 1.2 Q233F 1.4 Q233H 1.5Q233L 1.5 Q233M 1.5 Q233S 1.3 1.3 Q233T 1.4 T236A 1.3 1.3 T236C 1.3T236L 1.3 T236P 1.2 T236S 1.2 T236V 1.3 1.3 1.2 S237F 1.2 1.2 S237G 1.2S237H 1.2 S237L 1.3 S237M 1.2 S237N 1.2 S237R 1.3 1.3 S237V 1.3 D238A1.4 1.3 1.4 1.5 D238E 1.3 1.3 D238F 1.2 D238G 1.6 1.3 2.0 D238H 1.3 1.8D238M 1.4 D238P 1.4 1.2 1.0 D238R 1.5 1.2 D238S 1.3 1.3 1.0 D238V 1.71.2 1.9 D238W 1.3 L239F 1.2 L239G 1.2 L239S 1.7 1.3 L239Y 1.3 E240A 1.6T241M 1.3 T241V 1.4 1.3 S242A 1.2 1.2 1.3 S242C 1.4 S242G 1.3 S242I 1.21.0 S242L 1.4 S242M 1.3 S242P 1.4 1.4 2.0 S242R 1.2 1.4 S242T 1.2 1.31.7 S242V 1.2 1.3 S242Y 1.2 D243A 1.4 1.3 D243C 1.2 D243E 1.3 D243F 1.2D243G 1.2 D243L 1.4 1.4 1.3 D243M 1.2 1.2 D243R 1.2 1.1 D243T 1.4 D243V1.3 1.3 N246A 1.2 2.3 N246E 1.7 N246K 1.3 N246P 1.5 2.1 N246S 1.2 1.5N246T 1.3 N246V 2 S247A 1.3 S247E 1.5 S247L 1.2 1.9 S247P 1.9 S247T 1.21.1 S247Y 1.5 I248A 1.9 I248G 1.6 I248P 1.7 I248V 1.5 T252A 1.6 1.8T252D 1.6 T252E 1.2 T252G 1.5 1.8 T252N 1.4 1.6 T252Q 2.3 T252S 1.6 2.5T252V 1.2 1.5 L255A 1.5 L255C 1.3 1.2 L255E 1.2 L255M 1.2 1.2 L255Q 1.41.2 1.1 L255R 1.2 L255S 1.2 1.2 L255T 1.5 1.3 L255V 1.5 1.3 S259A 1.41.2 S259C 1.6 1.6 1.0 S259D 1.3 S259E 1.2 1.4 1.0 S259G 1.5 S259I 1.3S259K 1.3 S259L 1.5 1.5 S259M 1.4 1.4 1.4 S259Q 1.2 1.5 1.1 S259R 1.31.2 S259T 1.4 1.3 S259V 1.5 1.2 1.2 1.0 S259W 1.3 1.0 S259Y 1.3 1.5G262A 2.3 G262D 1.3 G262E 1.2 G262K 1.3 G262M 1.7 G262R 2.4 G262T 1.8G262V 1.5 N264C 1.2 N264G 1.2 N264I 1.3 1.5 1.0 N264L 1.2 N264M 1.3 1.3N264P 1.8 1.2 1.4 1.0 N264R 1.7 1.4 1.6 N264S 1.2 1.2 1.2 N264T 1.2L267A 1.2 1.5 1.0 L267E 1.2 1.6 L267H 1.3 1.2 L267K 1.4 L267M 1.5 L267Q1.6 L267S 1.6 L267T 1.4 1.1 L267W 1.3 T269A 1.3 1.0 T269D 1.4 T269E 1.61.3 T269G 1.3 1.4 1.0 T269H 1.4 T269K 1.5 T269L 1.4 1.3 1.0 T269M 1.31.3 1.1 T269N 1.5 1.5 T269Q 1.3 1.3 1.0 T269R 1.4 1.5 T269S 1.4 T269V1.2 1.4 T269Y 1.3

1. A lipase variant of a parent lipase, which variant has lipaseactivity, at least 75% but less than 100% sequence identity to SEQ IDNO: 3 and comprises a substitution at one or more positionscorresponding to positions 1; 2; 3; 4; 5; 6; 7; 9; 10; 11; 12; 16; 19;30; 31; 34; 36; 37; 39; 40; 42; 44; 51; 52; 53; 54; 56; 58; 59; 70; 71;72; 73; 83; 88; 92; 93; 95; 96; 100; 101; 102; 104; 106; 109; 110; 112;117; 119; 124; 125; 127; 128; 131; 132; 133; 134; 135; 137; 158; 159;160; 161; 162; 163; 165; 166; 167; 168; 170; 181; 182; 183; 189; 190;192; 194; 196; 202; 210; 211; 212; 220; 225; 227; 228; 229; 230; 231;233; 237; 238; 239; 240; 242; 246; 247; 248; 252; 259; 262; 264; 269 ofSEQ ID NO:
 3. 2. A lipase variant of a parent lipase, which variant haslipase activity, at least 75% but less than 100% sequence identity toSEQ ID NO: 3 and comprises a substitution at one or more positionscorresponding to positions 1(C,F,G,H,I,L,M,P,Q,R,V,W,Y); 2(A,C,D,M,N,P,Q,R,S,T,V); 3(A,E,G,K,Q,S,T,V); 4(A,P,Q,R,S,T); 5(E,K,S,T,V,Y);6(A,H,K,N,P,Q,R,S,V,W); 7(P); 8(C,G,H,N,P,Q,R,S,W,Y); 9(Y); 10(G,R,S,Y);11(G,H,I,L,P,Q,R,T); 12(A,I,K,L,M,N,R,S,T); 16(P,R,S,T,W);19(C,E,H,L,W,T); 30(A,F,H,I,L,N,P,S,T,V,W);31(A,C,D,E,F,G,H,I,K,P,Q,R,S,V); 34(A,G,V); 36(D,E,G,K,Q,R,S,V);37(A,E,G,S,V); 39(F,K,L,N,S); 40(A,D,E,G,I,L,R,V);41(A,D,E,G,H,K,L,P,Q,R,S,V,W); 42(A,E,G,K,L,M,R,S,T,V);44(A,C,E,G,H,K,M,P,Q,R,S,V,W); 51(A,D,G,H,K,L,M,V,Y); 52(L,V);53(A,D,G,H,I,L,M,P,Q,R,S,V); 54(A,D,E,G,I,K,L,S,V); 56(A,P,Q,R,T,V,W);58(C,G,I,P,R,S,V,W); 59(A,D,F,G,H,L,M,P,R,S,T,V); 61(G,M); 70(A,E,G,R);71(E,K,N,Q,R,W,Y); 72(A,C,D,G,K,R,S,T,V); 73(E,G,L,N,Q,R,S,T); 83(A);84(A,E,L,P,W,Y); 86(A,G,I,K,M,W); 88(M,Q,R,S); 90(D,F,G, L,S,T,V,W);91(E,F,G,Q,Y); 92(A,C,D,F,P,S,T,V,W); 93(A,I,R,V); 95(C,G,I,Q,R,S);96(A,C,D,E,G,N,Q,R,S,W,Y); 98(A,D,E,G,K,N,R,T,V,W);100(A,E,G,K,L,R,S,V,W); 101(D,H,R,S,V); 102(H,I,P,R,S);103(A,C,D,F,G,L,P,V); 104(A,C,D,E,K,L,M,P,Q,R,V,W,Y);106(A,C,G,L,P,Q,R,S,T,W); 109(A,D,E,G,H,L,N,P,Q,R,S,T); 110(C,P,S,V);112(F,H,Q,R,W); 113(M,W); 116(A,C,D,F,R,S,T); 117(A,D,G,N,P,R,S,V);119(D,E,F,I,R,S,T,V); 120(A,E,G,H, L,M,S,T,V,Y); 124(C,D,G,H,L,M,Q,R,S,V,W); 125(A,D,E,G,I,K,L,M,Q,R,S,V);127(A,F,G,H,I,Q,R,S,V,Y); 128(A,E,G,H,M,Q,S,T); 131(A,G,I,M,S,T,V,W);132(G,S,V); 133(A,E,K,L,M,R,V,W); 134(A,D,E,G,K,R,S,T,V);135(A,C,G,P,Q,R,T,V,W,Y); 137(A,F,G,P,R); 158(A,C,G,I,L,N,R,S,T,V,W);159(G,T); 160(A,D,G,I,L,Q,V); 161(C,G,I,K,L,P,Q,R,S);162(A,D,G,L,M,P,R,S,V,W); 163(A,D,E,H,L,M,Q,V,Y);165(A,C,G,K,L,M,P,R,V,W); 166(A,C,E,G,L,P,R,W); 167(C,F,H,I,L,M,R,V);168(A,C,D,G,R,S,V,W); 170(A,C,E,G,H,I,K,L,M,Q,R,S,V,W,Y);181(A,C,E,G,L,Q,R,S,V,W); 182(A,C,F,G,I,L,R,S,T,V,W,Y); 183(E,V);186(A,C,E,G,H,K,L,R,S,T,W); 189(A,C,G,L,M,P,R,S);190(A,E,G,I,K,N,R,V,W); 192(A,I,L,M,P,R,T,V); 194(A,D,E,F,G,H,L,Q,T,V);196(A,Q,Y); 201(D,G,L,P); 202(I,M); 204(E,K,L,P,R); 208(E,M,P,R,W);210(A,F,L,Q); 211(C,E,G,P,S,V,W); 212(D,E,G,H,K,N,P,Q,R,S,T,V,W);213(E,H,L,M,P,Q,R,W); 220(A,C,D,G,L,P,R,S,T,W); 225(A,L,M,P,Q,S);226(C,E,F,G,P,R,W,Y); 227(A,C,G,P,R,S,V,W); 228(A,C,D,G,L,P,R,V,W);229(A,I,L,N,R,S,T); 230(A,G,H,L,P,Q,R,S,T,V,W);231(A,C,G,H,K,R,S,V,W,Y); 233(A,F,H,L,M,S,T); 236(C,L,P,S,V);237(F,G,H,L,M,N,R,V); 238(A, E,F,G,H,M,P,R,S,V,W); 239(F,G,S,Y); 240(A);241(M,V); 242(A,C,G,I,L,M, P,R,T,V,Y); 243(A,C,E,F,G,M);246(A,E,K,P,S,T,V); 247(A,E,L,P,T,Y); 248(A,G,P,V);252(A,D,E,G,N,Q,S,V); 255(C,E,M,Q,R);259(A,C,D,E,G,I,K,L,M,Q,R,T,V,W,Y); 262(A,D,E,K,M,R,T,V);264(C,G,I,L,M,P,R,S,T); 267(E,H,K,M,Q,W);269(A,D,E,G,H,K,L,M,N,Q,R,S,V,Y) of SEQ ID NO:
 3. 3. The variant ofclaim 1 selected from the group consisting of: a. a polypeptide havingat least 80% but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3; b. a polypeptide encoded by a polynucleotide thathybridizes under low stringency conditions with (i) the polypeptidecoding sequence of SEQ ID NO: 1 or (ii) the full-length complement of(i); c. a polypeptide encoded by a polynucleotide having at least 80%,but less than 100% sequence identity to the polypeptide coding sequenceof SEQ ID NO: 3; and d. a fragment of the polypeptide of SEQ ID NO: 3,wherein the fragment has lipase activity.
 4. The variant of claim 1,wherein the variant has one or more improved property relative to theparent measured as an Improvement Factor (IF) that is greater than 1.0,wherein the improved property is selected from the group consisting of:Thermostability; Thermostability in Detergent; Hydrolytic Activity inDetergent; Stability in Detergent.
 5. The variant of claim 1, whereinthe Improvement Factor (IF) is greater than 1.0 in at least one of theassays selected from the group consisting of: A Thermostability Assay; BThermostability in Detergent Assay; C Performance Screening Assay; and DStability in Detergent Assay.
 6. The variant of claim 5, wherein theImprovement Factor (IF) is greater than 1.0 in at least two.
 7. Thevariant of claim 1, wherein the Improvement Factor (IF) is at least. 8.The variant of claim 1, wherein the number of substitutions are 1-40. 9.The variant of claim 1, which further comprises a substitution at one ormore positions corresponding to positions: ABT; Y28LC; I41T; C43S; D48E;K50E; Y6OAC; D61NKRAVLSTC; R80C; S84C; I86C; N87C; A90C; D91NKRAVLST;F94LTK; S98P; S103R; D113NKRAVLST; S114C; G116V; N120KD; K131F; G156D;N186Y; E201QKRAVLST; 1204VATSG; V205IL; L208VATSG; F213VATSG; H217N;D226NKRAVLST; T236A; T241S; D243NKRAVLSTH; V254IATSG; L255VATSG;D256NKRAVLST; L258VATSG; Y260WCGV; L267VATSG of SEQ ID NO:
 3. 10. Amethod of hydrolyzing a lipase substrate comprising contacting saidsubstrate with the variant of claim
 1. 11. A composition comprising thevariant of claim
 1. 12. A method of producing composition comprisingadding the variant of any of claims 1-9.
 13. A method of cleaningcomprising contacting a surface or an item with the cleaning compositionaccording to claim
 1. 14. A polynucleotide encoding the variant ofclaim
 1. 15. A nucleic acid construct comprising the polynucleotide ofclaim
 14. 16. An expression vector comprising the polynucleotide ofclaim
 14. 17. A host cell comprising the polynucleotide of claim
 14. 18.A method of producing a lipase variant, comprising: a. cultivating thehost cell of claim 17 under conditions suitable for expression of thevariant; and b. recovering the variant.
 19. A method for obtaining alipase variant and/or a fragment thereof, comprising introducing into aparent lipase a substitution at one or more positions corresponding topositions 1; 2; 3; 4; 5; 6; 7; 9; 10; 11; 12; 16; 19; 30; 31; 34; 36;37; 39; 40; 42; 44; 51; 52; 53; 54; 56; 58; 59; 70; 71; 72; 73; 83; 88;92; 93; 95; 96; 100; 101; 102; 104; 106; 109; 110; 112; 117; 119; 124;125; 127; 128; 131; 132; 133; 134; 135; 137; 158; 159; 160; 161; 162;163; 165; 166; 167; 168; 170; 181; 182; 183; 189; 190; 192; 194; 196;202; 210; 211; 212; 220; 225; 227; 228; 229; 230; 231; 233; 237; 238;239; 240; 242; 246; 247; 248; 252; 259; 262; 264; 269 of SEQ ID NO: 3,wherein the variant and/or fragment has lipase activity; and recoveringthe variant and/or fragment.